Modulators of ATP-binding cassette-transporters

ABSTRACT

Compounds of the present invention, and pharmaceutically acceptable compositions thereof, are useful as modulators of ATP-Binding Cassette (“ABC”) transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator (“CFTR”). The present invention also relates to methods of treating ABC transporter mediated diseases using compounds of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/271,088, filed Nov. 14, 2008 now U.S. Pat. No. 8,507,524, whichclaims the benefit under 35 U.S.C. §119 to U.S. provisional patentapplication Ser. No. 61/988,559 filed Nov. 16, 2007, the entire contentsof both applications are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of ATP-Binding Cassette(“ABC”) transporters or fragments thereof, including Cystic FibrosisTransmembrane Conductance Regulator (“CFTR”), compositions thereof, andmethods therewith. The present invention also relates to methods oftreating ABC transporter mediated diseases using such modulators.

BACKGROUND OF THE INVENTION

ABC transporters are a family of membrane transporter proteins thatregulate the transport of a wide variety of pharmacological agents,potentially toxic drugs, and xenobiotics, as well as anions. ABCtransporters are homologous membrane proteins that bind and use cellularadenosine triphosphate (ATP) for their specific activities. Some ofthese transporters were discovered as multi-drug resistance proteins(like the MDR1-P glycoprotein, or the multi-drug resistance protein,MRP1), defending malignant cancer cells against chemotherapeutic agents.To date, 48 ABC Transporters have been identified and grouped into 7families based on their sequence identity and function.

ABC transporters regulate a variety of important physiological roleswithin the body and provide defense against harmful environmentalcompounds. Because of this, they represent important potential drugtargets for the treatment of diseases associated with defects in thetransporter, prevention of drug transport out of the target cell, andintervention in other diseases in which modulation of ABC transporteractivity may be beneficial.

One member of the ABC transporter family commonly associated withdisease is the cAMP/ATP-mediated anion channel, CFTR. CFTR is expressedin a variety of cells types, including absorptive and secretoryepithelia cells, where it regulates anion flux across the membrane, aswell as the activity of other ion channels and proteins. In epitheliacells, normal functioning of CFTR is critical for the maintenance ofelectrolyte transport throughout the body, including respiratory anddigestive tissue. CFTR is composed of approximately 1480 amino acidsthat encode a protein made up of a tandem repeat of transmembranedomains, each containing six transmembrane helices and a nucleotidebinding domain. The two transmembrane domains are linked by a large,polar, regulatory (R)-domain with multiple phosphorylation sites thatregulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in CysticFibrosis (“CF”), the most common fatal genetic disease in humans. CysticFibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl− channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

In addition to Cystic Fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, chronic obstructivepulmonary disease (COPD), dry eye disease, and Sjögren's Syndrome.

COPD is characterized by airflow limitation that is progressive and notfully reversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such asCystic Fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)]. The diseases associated with the first class of ER malfunctionare Cystic fibrosis (due to misfolded ΔF508-CFTR as discussed above),Hereditary emphysema (due to a1-antitrypsin; non Piz variants),Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, suchas Protein C deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses (due to Lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-Hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus (due to Insulinreceptor), Laron dwarfism (due to Growth hormone receptor),Myleoperoxidase deficiency, Primary hypoparathyroidism (due toPreproparathyroid hormone), Melanoma (due to Tyrosinase). The diseasesassociated with the latter class of ER malfunction are Glycanosis CDGtype 1, Hereditary emphysema (due to α1-Antitrypsin (PiZ variant),Congenital hyperthyroidism, Osteogenesis imperfecta (due to Type I, II,IV procollagen), Hereditary hypofibrinogenemia (due to Fibrinogen), ACTdeficiency (due to α1-Antichymotrypsin), Diabetes insipidus (DI),Neurophyseal DI (due to Vasopvessin hormone/V2-receptor), Neprogenic DI(due to Aquaporin II), Charcot-Marie Tooth syndrome (due to Peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to PAPP and presenilins),Parkinson's disease, Amyotrophic lateral sclerosis, Progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, Spinocerebullar ataxia type I, Spinal andbulbar muscular atrophy, Dentatorubal pallidoluysian, and Myotonicdystrophy, as well as Spongiform encephalopathies, such as HereditaryCreutzfeldt-Jakob disease (due to Prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A) andStraussler-Scheinker syndrome (due to Prp processing defect).

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, impaired growth anddeath.

Acute and chronic diarrheas represent a major medical problem in manyareas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients ofacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). 16 million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Diarrhea in barn animals and pets such as cows, pigs, and horses, sheep,goats, cats and dogs, also known as scours, is a major cause of death inthese animals. Diarrhea can result from any major transition, such asweaning or physical movement, as well as in response to a variety ofbacterial or viral infections and generally occurs within the first fewhours of the animal's life.

The most common diarrhea causing bacteria is enterotoxogenic E-coli(ETEC) having the K99 pilus antigen. Common viral causes of diarrheainclude rotavirus and coronavirus. Other infectious agents includecryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include excretion of watery feces,dehydration and weakness. Coronavirus causes a more severe illness inthe newborn animals, and has a higher mortality rate than rotaviralinfection. Often, however, a young animal may be infected with more thanone virus or with a combination of viral and bacterial microorganisms atone time. This dramatically increases the severity of the disease.

Accordingly, there is a need for modulators of an ABC transporteractivity, and compositions thereof, that can be used to modulate theactivity of the ABC transporter in the cell membrane of a mammal.

There is a need for methods of treating ABC transporter mediateddiseases using such modulators of ABC transporter activity.

There is a need for methods of modulating an ABC transporter activity inan ex vivo cell membrane of a mammal.

There is a need for modulators of CFTR activity that can be used tomodulate the activity of CFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are useful asmodulators of ABC transporter activity. These compounds have the generalformula (I):

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R′₃,R₄, and n are described herein.

These compounds and pharmaceutically acceptable compositions are usefulfor treating or lessening the severity of a variety of diseases,disorders, or conditions, including, but not limited to, cysticfibrosis, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, DiabetesMellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, hereditaryemphysema, congenital hyperthyroidism, osteogenesis imperfecta,hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI),neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, spinocerebullar ataxia typeI, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian, andmyotonic dystrophy, as well as spongiform encephalopathies, such ashereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjogren'sdisease.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing,e.g. activity, by a measurable amount. Compounds that modulate ABCTransporter activity, such as CFTR activity, by increasing the activityof the ABC Transporter, e.g., a CFTR anion channel, are called agonists.Compounds that modulate ABC Transporter activity, such as CFTR activity,by decreasing the activity of the ABC Transporter, e.g., CFTR anionchannel, are called antagonists. An agonist interacts with an ABCTransporter, such as CFTR anion channel, to increase the ability of thereceptor to transduce an intracellular signal in response to endogenousligand binding. An antagonist interacts with an ABC Transporter, such asCFTR, and competes with the endogenous ligand(s) or substrate(s) forbinding site(s) on the receptor to decrease the ability of the receptorto transduce an intracellular signal in response to endogenous ligandbinding.

The phrase “treating or reducing the severity of an ABC Transportermediated disease” refers both to treatments for diseases that aredirectly caused by ABC Transporter and/or CFTR activities andalleviation of symptoms of diseases not directly caused by ABCTransporter and/or CFTR anion channel activities. Examples of diseaseswhose symptoms may be affected by ABC Transporter and/or CFTR activityinclude, but are not limited to, Cystic fibrosis, Hereditary emphysema,Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, suchas Protein C deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, Hereditary emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders asuch as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eyedisease, and Sjogren's disease.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausolito: 1999, and “March'sAdvanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J.,John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J.,John Wiley & Sons, New York: 2001.

As used herein the term “aliphatic’ encompasses the terms alkyl,alkenyl, alkynyl, each of which being optionally substituted as setforth below.

As used herein, an “alkyl” group refers to a saturated aliphatichydrocarbon group containing 1-8 (e.g., 1-6 or 1-4) carbon atoms. Analkyl group can be straight or branched. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or2-ethylhexyl. An alkyl group can be substituted (i.e., optionallysubstituted) with one or more substituents such as halo, cycloaliphatic[e.g., cycloalkyl or cycloalkenyl], heterocycloaliphatic [e.g.,heterocycloalkyl or heterocycloalkenyl], aryl, heteroaryl, alkoxy,aroyl, heteroaroyl, acyl [e.g., (aliphatic)carbonyl,(cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl], nitro,cyano, amido [e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino,heteroaralkylcarbonylamino], amino [e.g., aliphaticamino,cycloaliphaticamino, or heterocycloaliphaticamino], sulfonyl [e.g.,aliphaticsulfonyl], sulfinyl, sulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy,heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy,heteroarylalkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy. Withoutlimitation, some examples of substituted alkyls include carboxyalkyl(such as HOOC-alkyl, alkoxycarbonylalkyl, and alkylcarbonyloxyalkyl),cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, hydroxyalkyl, aralkyl,(alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as(alkylsulfonylamino)alkyl), aminoalkyl, amidoalkyl,(cycloaliphatic)alkyl, cyanoalkyl, or haloalkyl.

As used herein, an “alkenyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and at least onedouble bond. Like an alkyl group, an alkenyl group can be straight orbranched. Examples of an alkenyl group include, but are not limited to,allyl, isoprenyl, 2-butenyl, and 2-hexenyl. An alkenyl group can beoptionally substituted with one or more substituents such as halo,cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl,heteroaroyl, acyl [e.g., (cycloaliphatic)carbonyl, or(heterocycloaliphatic)carbonyl], nitro, cyano, acyl [e.g.,aliphaticcarbonyl, cycloaliphaticcarbonyl, arylcarbonyl,heterocycloaliphaticcarbonyl or heteroarylcarbonyl], amido [e.g.,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,arylaminocarbonyl, or heteroarylaminocarbonyl], amino [e.g.,aliphaticamino, or aliphaticsulfonylamino], sulfonyl [e.g.,alkylsulfonyl, cycloaliphaticsulfonyl, or arylsulfonyl], sulfinyl,sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy,carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy,heteroaryloxy, aralkyloxy, heteroarylalkoxy, alkoxycarbonyl,alkylcarbonyloxy, or hydroxy.

As used herein, an “alkynyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and has at least onetriple bond. An alkynyl group can be straight or branched. Examples ofan alkynyl group include, but are not limited to, propargyl and butynyl.An alkynyl group can be optionally substituted with one or moresubstituents such as aroyl, heteroaroyl, alkoxy, cycloalkyloxy,heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, nitro, carboxy,cyano, halo, hydroxy, sulfo, mercapto, sulfanyl [e.g., aliphaticsulfanylor cycloaliphaticsulfanyl], sulfinyl [e.g., aliphaticsulfinyl orcycloaliphaticsulfinyl], sulfonyl [e.g., aliphaticsulfonyl,aliphaticaminosulfonyl, or cycloaliphaticsulfonyl], amido [e.g.,aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,cycloalkylcarbonylamino, arylaminocarbonyl, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(cycloalkylalkyl)carbonylamino, heteroaralkylcarbonylamino,heteroarylcarbonylamino or heteroarylaminocarbonyl], urea, thiourea,sulfamoyl, sulfamide, alkoxycarbonyl, alkylcarbonyloxy, cycloaliphatic,heterocycloaliphatic, aryl, heteroaryl, acyl [e.g.,(cycloaliphatic)carbonyl or (heterocycloaliphatic)carbonyl], amino[e.g., aliphaticamino], sulfoxy, oxo, carboxy, carbamoyl,(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, or (heteroaryl)alkoxy.

As used herein, an “amido” encompasses both “aminocarbonyl” and“carbonylamino” These terms when used alone or in connection withanother group refers to an amido group such as N(R^(X)R^(Y))—C(O)— orR^(Y)C(O)—N(R^(X))— when used terminally and —C(O)—N(R^(X))— or—N(R^(X))—C(O)— when used internally, wherein R^(X) and R^(Y) aredefined below. Examples of amido groups include alkylamido (such asalkylcarbonylamino or alkylcarbonylamino), (heterocycloaliphatic)amido,(heteroaralkyl)amido, (heteroaryl)amido, (heterocycloalkyl)alkylamido,arylamido, aralkylamido, (cycloalkyl)alkylamido, or cycloalkylamido.

As used herein, an “amino” group refers to —NR^(X)R^(Y) wherein each ofR^(X) and R^(Y) is independently hydrogen, alkyl, cycloaliphatic,(cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic,(heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl,sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl,((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or(heteroaraliphatic)carbonyl, each of which being defined herein andbeing optionally substituted. Examples of amino groups includealkylamino, dialkylamino, or arylamino When the term “amino” is not theterminal group (e.g., alkylcarbonylamino), it is represented by—NR^(X)—. R^(X) has the same meaning as defined above.

As used herein, an “aryl” group used alone or as part of a larger moietyas in “aralkyl”, “aralkoxy”, or “aryloxyalkyl” refers to monocyclic(e.g., phenyl); bicyclic (e.g., indenyl, naphthalenyl,tetrahydronaphthyl, tetrahydroindenyl); and tricyclic (e.g., fluorenyltetrahydrofluorenyl, or tetrahydroanthracenyl, anthracenyl) ring systemsin which the monocyclic ring system is aromatic or at least one of therings in a bicyclic or tricyclic ring system is aromatic. The bicyclicand tricyclic ring systems include benzofused 2-3 membered carbocyclicrings. For example, a benzofused group includes phenyl fused with two ormore C₄₋₈ carbocyclic moieties. An aryl is optionally substituted withone or more substituents including aliphatic [e.g., alkyl, alkenyl, oralkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic;heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl;alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy;heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl;heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring of abenzofused bicyclic or tricyclic aryl); nitro; carboxy; amido; acyl[e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl;((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl;(heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl oraminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl orcycloaliphaticsulfinyl]; sulfanyl [e.g., aliphaticsulfanyl]; cyano;halo; hydroxy; mercapto; sulfoxy; urea; thiourea; sulfamoyl; sulfamide;or carbamoyl. Alternatively, an aryl can be unsubstituted.

Non-limiting examples of substituted aryls include haloaryl [e.g.,mono-, di (such as p,m-dihaloaryl), and (trihalo)aryl]; (carboxy)aryl[e.g., (alkoxycarbonyl)aryl, ((aralkyl)carbonyloxy)aryl, and(alkoxycarbonyl)aryl]; (amido)aryl [e.g., (aminocarbonyl)aryl,(((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl,(arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl];aminoaryl [e.g., ((alkylsulfonyl)amino)aryl or ((dialkyl)amino)aryl];(cyanoalkyl)aryl; (alkoxy)aryl; (sulfamoyl)aryl [e.g.,(aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl;(hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxy)aryl,((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl; (nitroalkyl)aryl;(((alkylsulfonyl)amino)alkyl)aryl; ((heterocycloaliphatic)carbonyl)aryl;((alkylsulfonyl)alkyl)aryl; (cyanoalkyl)aryl; (hydroxyalkyl)aryl;(alkylcarbonyl)aryl; alkylaryl; (trihaloalkyl)aryl;p-amino-m-alkoxycarbonylaryl; p-amino-m-cyanoaryl; p-halo-m-aminoaryl;or (m-(heterocycloaliphatic)-o-(alkyl))aryl.

As used herein, an “araliphatic” such as an “aralkyl” group refers to analiphatic group (e.g., a C₁₋₄ alkyl group) that is substituted with anaryl group. “Aliphatic,” “alkyl,” and “aryl” are defined herein. Anexample of an araliphatic such as an aralkyl group is benzyl.

As used herein, an “aralkyl” group refers to an alkyl group (e.g., aC₁₋₄ alkyl group) that is substituted with an aryl group. Both “alkyl”and “aryl” have been defined above. An example of an aralkyl group isbenzyl. An aralkyl is optionally substituted with one or moresubstituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl,including carboxyalkyl, hydroxyalkyl, or haloalkyl such astrifluoromethyl], cycloaliphatic [e.g., cycloalkyl or cycloalkenyl],(cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl,heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy,heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro,carboxy, alkoxycarbonyl, alkylcarbonyloxy, amido [e.g., aminocarbonyl,alkylcarbonylamino, cycloalkylcarbonylamino,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, or heteroaralkylcarbonylamino], cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, a “bicyclic ring system” includes 8-12 (e.g., 9, 10, or11) membered structures that form two rings, wherein the two rings haveat least one atom in common (e.g., 2 atoms in common). Bicyclic ringsystems include bicycloaliphatics (e.g., bicycloalkyl orbicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclicheteroaryls.

As used herein, a “cycloaliphatic” group encompasses a “cycloalkyl”group and a “cycloalkenyl” group, each of which being optionallysubstituted as set forth below.

As used herein, a “cycloalkyl” group refers to a saturated carbocyclicmono- or bicyclic (fused or bridged) ring of 3-10 (e.g., 5-10) carbonatoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, cubyl,octahydro-indenyl, decahydro-naphthyl, bicyclo[3.2.1]octyl,bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2.]decyl,bicyclo[2.2.2]octyl, adamantyl, azacycloalkyl, or((aminocarbonyl)cycloalkyl)cycloalkyl. A “cycloalkenyl” group, as usedherein, refers to a non-aromatic carbocyclic ring of 3-10 (e.g., 4-8)carbon atoms having one or more double bonds. Examples of cycloalkenylgroups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl,cyclooctenyl, hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl,cyclopentenyl, bicyclo[2.2.2]octenyl, or bicyclo[3.3.1]nonenyl. Acycloalkyl or cycloalkenyl group can be optionally substituted with oneor more substituents such as aliphatic [e.g., alkyl, alkenyl, oralkynyl], cycloaliphatic, (cycloaliphatic) aliphatic,heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl,heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy,aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,(cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino,(aryl)carbonylamino, (araliphatic)carbonylamino,(heterocycloaliphatic)carbonylamino,((heterocycloaliphatic)aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], cyano, halo, hydroxy, mercapto, sulfonyl[e.g., alkylsulfonyl and arylsulfonyl], sulfinyl [e.g., alkylsulfinyl],sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea, sulfamoyl,sulfamide, oxo, or carbamoyl.

As used herein, “cyclic moiety” includes cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl, each of which has beendefined previously.

As used herein, the term “heterocycloaliphatic” encompasses aheterocycloalkyl group and a heterocycloalkenyl group, each of whichbeing optionally substituted as set forth below.

As used herein, a “heterocycloalkyl” group refers to a 3-10 memberedmono- or bicylic (fused or bridged) (e.g., 5- to 10-membered mono- orbicyclic) saturated ring structure, in which one or more of the ringatoms is a heteroatom (e.g., N, O, S, or combinations thereof). Examplesof a heterocycloalkyl group include piperidyl, piperazyl,tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl,1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl,octahydrobenzofuryl, octahydrochromenyl, octahydrothiochromenyl,octahydroindolyl, octahydropyrindinyl, decahydroquinolinyl,octahydrobenzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl,1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and2,6-dioxa-tricyclo[3.3.1.0^(3,7)]nonyl. A monocyclic heterocycloalkylgroup can be fused with a phenyl moiety such as tetrahydroisoquinoline.A “heterocycloalkenyl” group, as used herein, refers to a mono- orbicylic (e.g., 5- to 10-membered mono- or bicyclic) non-aromatic ringstructure having one or more double bonds, and wherein one or more ofthe ring atoms is a heteroatom (e.g., N, O, or S). Monocyclic andbicycloheteroaliphatics are numbered according to standard chemicalnomenclature.

A heterocycloalkyl or heterocycloalkenyl group can be optionallysubstituted with one or more substituents such as aliphatic [e.g.,alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic)aliphatic,heterocycloaliphatic, (heterocycloaliphatic)aliphatic, aryl, heteroaryl,alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy,heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,(cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino,(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,((heterocycloaliphatic) aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy, mercapto,sulfonyl [e.g., alkylsulfonyl or arylsulfonyl], sulfinyl [e.g.,alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

A “heteroaryl” group, as used herein, refers to a monocyclic, bicyclic,or tricyclic ring system having 4 to 15 ring atoms wherein one or moreof the ring atoms is a heteroatom (e.g., N, O, S, or combinationsthereof) and in which the monocyclic ring system is aromatic or at leastone of the rings in the bicyclic or tricyclic ring systems is aromatic.A heteroaryl group includes a benzofused ring system having 2 to 3rings. For example, a benzofused group includes benzo fused with one ortwo 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl,indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl,benzo[b]thiophenyl, quinolinyl, or isoquinolinyl). Some examples ofheteroaryl are azetidinyl, pyridyl, 1H-indazolyl, furyl, pyrrolyl,thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl,isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine,dihydroindole, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thiophenyl,indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl,quinazolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl,4H-quinolizyl, benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.

Without limitation, monocyclic heteroaryls include furyl, thiophenyl,2H-pyrrolyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pyranyl,pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl.Monocyclic heteroaryls are numbered according to standard chemicalnomenclature.

Without limitation, bicyclic heteroaryls include indolizyl, indolyl,isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl,quinolinyl, isoquinolinyl, indolizyl, isoindolyl, indolyl,benzo[b]furyl, bexo[b]thiophenyl, indazolyl, benzimidazyl,benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl,phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl.Bicyclic heteroaryls are numbered according to standard chemicalnomenclature.

A heteroaryl is optionally substituted with one or more substituentssuch as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic;(cycloaliphatic)aliphatic; heterocycloaliphatic;(heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy;(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy;(araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo(on a non-aromatic carbocyclic or heterocyclic ring of a bicyclic ortricyclic heteroaryl); carboxy; amido; acyl [e.g., aliphaticcarbonyl;(cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl;(araliphatic)carbonyl; (heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl oraminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g.,aliphaticsulfanyl]; nitro; cyano; halo; hydroxy; mercapto; sulfoxy;urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, aheteroaryl can be unsubstituted.

Non-limiting examples of substituted heteroaryls include(halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl];(carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl]; cyanoheteroaryl;aminoheteroaryl [e.g., ((alkylsulfonyl)amino)heteroaryl and((dialkyl)amino)heteroaryl]; (amido)heteroaryl [e.g.,aminocarbonylheteroaryl, ((alkylcarbonyl)amino)heteroaryl,((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl,(((heteroaryl)amino)carbonyl)heteroaryl,((heterocycloaliphatic)carbonyl)heteroaryl, and((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl;(alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g.,(aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g.,(alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl;(alkoxyalkyl)heteroaryl; (hydroxy)heteroaryl;((carboxy)alkyl)heteroaryl; [((dialkyl)amino)alkyl]heteroaryl;(heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl;(nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl;((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl;(acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl]; (alkyl)heteroaryl,and (haloalkyl)heteroaryl [e.g., trihaloalkylheteroaryl].

A “heteroaraliphatic” (such as a heteroaralkyl group) as used herein,refers to an aliphatic group (e.g., a C₁₋₄ alkyl group) that issubstituted with a heteroaryl group. “Aliphatic,” “alkyl,” and“heteroaryl” have been defined above.

A “heteroaralkyl” group, as used herein, refers to an alkyl group (e.g.,a C₁₋₄ alkyl group) that is substituted with a heteroaryl group. Both“alkyl” and “heteroaryl” have been defined above. A heteroaralkyl isoptionally substituted with one or more substituents such as alkyl(including carboxyalkyl, hydroxyalkyl, and haloalkyl such astrifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl,heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy,cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl,alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino,cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino,arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, “cyclic moiety” includes cycloalkyl, heterocycloalkyl,cycloalkenyl, heterocycloalkenyl, aryl, or heteroaryl, each of which hasbeen defined previously.

As used herein, an “acyl” group refers to a formyl group or R^(X)—C(O)—(such as -alkyl-C(O)—, also referred to as “alkylcarbonyl”) where R^(X)and “alkyl” have been defined previously. Acetyl and pivaloyl areexamples of acyl groups.

As used herein, an “aroyl” or “heteroaroyl” refers to an aryl-C(O)— or aheteroaryl-C(O)—. The aryl and heteroaryl portion of the aroyl orheteroaroyl is optionally substituted as previously defined.

As used herein, an “alkoxy” group refers to an alkyl-O— group where“alkyl” has been defined previously.

As used herein, a “carbamoyl” group refers to a group having thestructure —O—CO—NR^(X)R^(Y) or —NR^(X)—CO—O—R^(Z) wherein R^(X) andR^(Y) have been defined above and R^(Z) can be aliphatic, aryl,araliphatic, heterocycloaliphatic, heteroaryl, or heteroaraliphatic.

As used herein, a “carboxy” group refers to —COOH, —COOR^(X), —OC(O)H,—OC(O)R^(X) when used as a terminal group; or —OC(O)— or —C(O)O— whenused as an internal group.

As used herein, a “haloaliphatic” group refers to an aliphatic groupsubstituted with 1, 2, or 3 halogen. For instance, the term haloalkylincludes the group —CF₃.

As used herein, a “mercapto” group refers to —SH.

As used herein, a “sulfo” group refers to —SO₃H or —SO₃R^(X) when usedterminally or —S(O)₃— when used internally.

As used herein, a “sulfamide” group refers to the structure—NR^(X)—S(O)₂—NR^(Y)R^(Z) when used terminally and —NR^(X)—S(O)₂—NR^(Y)—when used internally, wherein R^(X), R^(Y), and R^(Z) have been definedabove.

As used herein, a “sulfamoyl” group refers to the structure—S(O)₂—NR^(X)R^(Y) or —NR^(X)—S(O)₂—R^(Z) when used terminally; or—S(O)₂—NR^(X)— or —NR^(X)—S(O)₂— when used internally, wherein R^(X),R^(Y), and R^(Z) are defined above.

As used herein a “sulfanyl” group refers to —S—R^(X) when usedterminally and —S— when used internally, wherein R^(X) has been definedabove. Examples of sulfanyls include alkylsulfanyl.

As used herein a “sulfinyl” group refers to —S(O)—R^(X) when usedterminally and —S(O)— when used internally, wherein R^(X) has beendefined above.

As used herein, a “sulfonyl” group refers to —S(O)₂—R^(X) when usedterminally and —S(O)₂— when used internally, wherein R^(X) has beendefined above.

As used herein, a “sulfoxy” group refers to —O—SO—R^(X) or —SO—O—R^(X),when used terminally and —O—S(O)— or —S(O)—O— when used internally,where R^(X) has been defined above.

As used herein, a “halogen” or “halo” group refers to fluorine,chlorine, bromine or iodine.

As used herein, an “alkoxycarbonyl,” which is encompassed by the termcarboxy, used alone or in connection with another group refers to agroup such as alkyl-O—C(O)—.

As used herein, an “alkoxyalkyl” refers to an alkyl group such asalkyl-O-alkyl-, wherein alkyl has been defined above.

As used herein, a “carbonyl” refer to —C(O)—.

As used herein, an “oxo” refers to ═O.

As used herein, an “aminoalkyl” refers to the structure(R^(X)R^(Y))N-alkyl-.

As used herein, a “cyanoalkyl” refers to the structure (NC)-alkyl-.

As used herein, a “urea” group refers to the structure—NR^(X)—CO—NR^(Y)R^(Z) and a “thiourea” group refers to the structure—NR^(X)—CS—NR^(Y)R^(Z) when used terminally and —NR^(X)—CO—NR^(Y)— or—NR^(X)—CS—NR^(Y)— when used internally, wherein R^(X), R^(Y), and R^(Z)have been defined above.

As used herein, a “guanidino” group refers to the structure —N═C(N(R^(X)R^(Y)))N(R^(X)R^(Y)) wherein R^(X) and R^(Y) have been defined above.

As used herein, the term “amidino” group refers to the structure—C═(NR^(X))N(R^(X)R^(Y)) wherein R^(X) and R^(Y) have been definedabove.

In general, the term “vicinal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to adjacent carbon atoms.

In general, the term “geminal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to the same carbon atom.

The terms “terminally” and “internally” refer to the location of a groupwithin a substituent. A group is terminal when the group is present atthe end of the substituent not further bonded to the rest of thechemical structure. Carboxyalkyl, i.e., R^(X)O(O)C-alkyl is an exampleof a carboxy group used terminally. A group is internal when the groupis present in the middle of a substituent to at the end of thesubstituent bound to the rest of the chemical structure. Alkylcarboxy(e.g., alkyl-C(O)O— or alkyl-OC(O)—) and alkylcarboxyaryl (e.g.,alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxy groupsused internally.

As used herein, the term “amidino” group refers to the structure—C═(NR^(X))N(R^(X)R^(Y)) wherein R^(X) and e have been defined above.

As used herein, “cyclic group” includes mono-, bi-, and tri-cyclic ringsystems including cycloaliphatic, heterocycloaliphatic, aryl, orheteroaryl, each of which has been previously defined.

As used herein, a “bridged bicyclic ring system” refers to a bicyclicheterocyclicalipahtic ring system or bicyclic cycloaliphatic ring systemin which the rings are bridged. Examples of bridged bicyclic ringsystems include, but are not limited to, adamantanyl, norbornanyl,bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl,bicyclo[3.2.3]nonyl, 2-oxa-bicyclo[2.2.2]octyl,1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and2,6-dioxa-tricyclo[3.3.1.03,7]nonyl. A bridged bicyclic ring system canbe optionally substituted with one or more substituents such as alkyl(including carboxyalkyl, hydroxyalkyl, and haloalkyl such astrifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl,heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy,cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl,alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino,cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino,arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, an “aliphatic chain” refers to a branched or straightaliphatic group (e.g., alkyl groups, alkenyl groups, or alkynyl groups).A straight aliphatic chain has the structure —[CH₂]_(v)—, where v is1-6. A branched aliphatic chain is a straight aliphatic chain that issubstituted with one or more aliphatic groups. A branched aliphaticchain has the structure —[CHQ]_(v)— where Q is hydrogen or an aliphaticgroup; however, Q shall be an aliphatic group in at least one instance.The term aliphatic chain includes alkyl chains, alkenyl chains, andalkynyl chains, where alkyl, alkenyl, and alkynyl are defined above.

The phrase “optionally substituted” is used interchangeably with thephrase “substituted or unsubstituted.” As described herein, compounds ofthe invention can optionally be substituted with one or moresubstituents, such as are illustrated generally above, or as exemplifiedby particular classes, subclasses, and species of the invention. Asdescribed herein, the variables R₁, R₂, R₃, and R₄, and other variablescontained therein formulae I encompass specific groups, such as alkyland aryl. Unless otherwise noted, each of the specific groups for thevariables R₁, R₂, R₃, and R₄, and other variables contained therein canbe optionally substituted with one or more substituents describedherein. Each substituent of a specific group is further optionallysubstituted with one to three of halo, cyano, oxoalkoxy, hydroxy, amino,nitro, aryl, haloalkyl, and alkyl. For instance, an alkyl group can besubstituted with alkylsulfanyl and the alkylsulfanyl can be optionallysubstituted with one to three of halo, cyano, oxoalkoxy, hydroxy, amino,nitro, aryl, haloalkyl, and alkyl. As an additional example, thecycloalkyl portion of a (cycloalkyl)carbonylamino can be optionallysubstituted with one to three of halo, cyano, alkoxy, hydroxy, nitro,haloalkyl, and alkyl. When two alkoxy groups are bound to the same atomor adjacent atoms, the two alkoxy groups can form a ring together withthe atom(s) to which they are bound.

In general, the term “substituted,” whether preceded by the term“optionally” or not, refers to the replacement of hydrogen radicals in agiven structure with the radical of a specified substituent. Specificsubstituents are described above in the definitions and below in thedescription of compounds and examples thereof. Unless otherwiseindicated, an optionally substituted group can have a substituent ateach substitutable position of the group, and when more than oneposition in any given structure can be substituted with more than onesubstituent selected from a specified group, the substituent can beeither the same or different at every position. A ring substituent, suchas a heterocycloalkyl, can be bound to another ring, such as acycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings shareone common atom. As one of ordinary skill in the art will recognize,combinations of substituents envisioned by this invention are thosecombinations that result in the formation of stable or chemicallyfeasible compounds.

The phrase “up to”, as used herein, refers to zero or any integer numberthat is equal or less than the number following the phrase. For example,“up to 3” means any one of 0, 1, 2, and 3.

The phrase “stable or chemically feasible,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

As used herein, an effective amount is defined as the amount required toconfer a therapeutic effect on the treated patient, and is typicallydetermined based on age, surface area, weight, and condition of thepatient. The interrelationship of dosages for animals and humans (basedon milligrams per meter squared of body surface) is described byFreireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surfacearea may be approximately determined from height and weight of thepatient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley,N.Y., 537 (1970). As used herein, “patient” refers to a mammal,including a human.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. Additionally, unless otherwise stated, structuresdepicted herein are also meant to include compounds that differ only inthe presence of one or more isotopically enriched atoms. For example,compounds having the present structures except for the replacement ofhydrogen by deuterium or tritium, or the replacement of a carbon by a¹³C— or ¹⁴C-enriched carbon are within the scope of this invention. Suchcompounds are useful, for example, as analytical tools or probes inbiological assays.

When a ring substituent is depicted as in the following example, it isunderstood that it may be a substituent on any ring position as valencyallows and not just the ring the connector line is drawn to. Forexample, in

R₁ may be in any available position on rings A and/or B.

Compounds

Compounds of the present invention are useful modulators of ABCtransporters and are useful in the treatment of ABC transport mediateddiseases.

A. Generic Compounds

The present invention includes a compound of formula (I),

or a pharmaceutically acceptable salt thereof, wherein:

each R₁ is an optionally substituted C₁₋₆ aliphatic, an optionallysubstituted aryl, an optionally substituted heteroaryl, an optionallysubstituted C₃₋₁₀ cycloaliphatic, an optionally substituted 3 to 10membered heterocycloaliphatic, carboxy [e.g., hydroxycarbonyl oralkoxycarbonyl], amido [e.g., aminocarbonyl], amino, halo, alkoxy,cyano, or hydroxy;

provided that at least one R₁ is an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic, anoptionally substituted aryl, or an optionally substituted heteroarylattached to the 1-position of the isoquinoline ring;

R₂ is hydrogen, an optionally substituted C₁₋₆ aliphatic, an optionallysubstituted C₃₋₆ cycloaliphatic, an optionally substituted phenyl, or anoptionally substituted heteroaryl;

R₃ and R′₃ together with the carbon atom to which they are attached forman optionally substituted C₃₋₂ cycloaliphatic or an optionallysubstituted heterocycloaliphatic;

R₄ is an optionally substituted aryl or an optionally substitutedheteroaryl; and

n is 1, 2, 3, 4, 5, or 6.

Specific Embodiments

A. Substituent R₁

Each R₁ is independently an optionally substituted C₁₋₆ aliphatic, anoptionally substituted aryl, an optionally substituted heteroaryl, anoptionally substituted C₃₋₁₀ membered cycloaliphatic, an optionallysubstituted 3 to 10 membered heterocycloaliphatic, carboxy [e.g.,hydroxycarbonyl or alkoxycarbonyl], amido [e.g., aminocarbonyl], amino,halo, cyano or hydroxy.

In some embodiments, one R₁ is an optionally substituted C₁₋₆ aliphatic.In several examples, one R₁ is an optionally substituted C₁₋₆ alkyl, anoptionally substituted C₂₋₆ alkenyl, or an optionally substituted C₂₋₆alkynyl. In several examples, one R₁ is C₁₋₆ alkyl, C₂₋₆ alkenyl, orC₂₋₆ alkynyl.

In several embodiments, one R₁ is an aryl or heteroaryl with 1, 2, or 3substituents. In several examples, one R₁ is a monocyclic aryl orheteroaryl. In several embodiments, R₁ is an aryl or heteroaryl with 1,2, or 3 substituents. In several examples, R₁ is a monocyclic aryl orheteroaryl.

In several embodiments, at least one R₁ is an optionally substitutedaryl or an optionally substituted heteroaryl and R₁ is bonded to thecore structure at the 1 position on the isoquinoline ring.

In several embodiments, one R₁ is phenyl with up to 3 substituents. Inseveral embodiments, R₁ is phenyl with up to 3 substituents.

In several embodiments, one R₁ is a heteroaryl ring with up to 3substituents. In certain embodiments, one R₁ is a monocyclic heteroarylring with up to 3 substituents. In other embodiments, one R₁ is abicyclic heteroaryl ring with up to 3 substituents. In severalembodiments, R₁ is a heteroaryl ring with up to 3 substituents. Incertain embodiments, R₁ is a monocyclic heteroaryl ring with up to 3substituents. In other embodiments, R₁ is a bicyclic heteroaryl ringwith up to 3 substituents.

In several embodiments, one R₁ is carboxy [e.g., hydroxycarbonyl oralkoxycarbonyl]. Or, one R₁ is amido [e.g., aminocarbonyl]. Or, one R₁is amino Or, is halo. Or, is cyano. Or, hydroxyl.

In some embodiments, R₁ is hydrogen, methyl, ethyl, i-propyl, t-butyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, allyl, F, Cl, methoxy,ethoxy, i-propoxy, t-butoxy, CF₃, OCF₃, CN, hydroxyl, or amino. Inseveral examples, R₁ is hydrogen, methyl, methoxy, F, CF₃ or OCF₃. Inseveral examples, R₁ can be hydrogen. Or, R₁ can be methyl. Or, R₁ canbe CF₃. Or, R₁ can be methoxy.

In several embodiments, R₁ is substituted with no more than threesubstituents selected from halo, oxo, or optionally substitutedaliphatic, cycloaliphatic, heterocycloaliphatic, amino [e.g.,(aliphatic)amino], amido [e.g., aminocarbonyl,((aliphatic)amino)carbonyl, and ((aliphatic)₂-amino)carbonyl], carboxy[e.g., alkoxycarbonyl and hydroxycarbonyl], sulfamoyl [e.g.,aminosulfonyl, ((aliphatic)₂-amino)sulfonyl,((cycloaliphatic)aliphatic)aminosulfonyl, and((cycloaliphatic)amino)sulfonyl], cyano, alkoxy, aryl, heteroaryl [e.g.,monocyclic heteroaryl and bicycloheteroaryl], sulfonyl [e.g.,aliphaticsulfonyl or (heterocycloaliphatic)sulfonyl], sulfinyl [e.g.,aliphaticsulfinyl], aroyl, heteroaroyl, or heterocycloaliphaticcarbonyl.

In several embodiments, R₁ is substituted with halo. Examples of R₁substituents include F, Cl, and Br. In several examples, R₁ issubstituted with F.

In several embodiments, R₁ is substituted with an optionally substitutedaliphatic. Examples of R₁ substituents include optionally substitutedalkoxyaliphatic, heterocycloaliphatic, aminoalkyl, hydroxyalkyl,(heterocycloalkyl)aliphatic, alkylsulfonylaliphatic,alkylsulfonylaminoaliphatic, alkylcarbonylaminoaliphatic,alkylaminoaliphatic, or alkylcarbonylaliphatic.

In several embodiments, R₁ is substituted with an optionally substitutedamino Examples of R₁ substituents include aliphaticcarbonylamino,aliphaticamino, arylamino, or aliphaticsulfonylamino.

In several embodiments, R₁ is substituted with a sulfonyl. Examples ofR₁ substituents include heterocycloaliphaticsulfonyl, aliphaticsulfonyl, aliphaticaminosulfonyl, aminosulfonyl,aliphaticcarbonylaminosulfonyl, alkoxyalkylheterocycloalkylsulfonyl,alkylheterocycloalkylsulfonyl, alkylaminosulfonyl,cycloalkylaminosulfonyl, (heterocycloalkyl)alkylaminosulfonyl, andheterocycloalkylsulfonyl.

In several embodiments, R₁ is substituted with carboxy. Examples of R₁substituents include alkoxycarbonyl and hydroxycarbonyl.

In several embodiments R₁ is substituted with amido. Examples of R₁substituents include alkylaminocarbonyl, aminocarbonyl,((aliphatic)₂-amino)carbonyl, and[((aliphatic)aminoaliphatic)amino]carbonyl.

In several embodiments, R₁ is substituted with carbonyl. Examples of R₁substituents include arylcarbonyl, cycloaliphaticcarbonyl,heterocycloaliphaticcarbonyl, and heteroarylcarbonyl.

In some embodiments, R₁ is hydrogen. In some embodiments, R₁ is—Z^(A)R₅, wherein each Z^(A) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(A) are optionally and independently replaced by —CO—,—CS—, —CONR^(A)—, —CONR^(A)NR^(A)—, —CO₂—, —OCO—, —NR^(A)CO₂—, —O—,—NR^(A)CONR^(A)—, —OCONR^(A)—, —NR^(A)NR^(A)—, —NR^(A)CO—, —S—, —SO—,—SO₂—, —NR^(A)—, —SO₂NR^(A)—, —NR^(A)SO₂—, or —NR^(A)SO₂NR^(A)—. Each R₅is independently R^(A), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃. EachR^(A) is independently a hydrogen, C₁₋₈ aliphatic group, acycloaliphatic, a heterocycloaliphatic, an aryl, or a heteroaryl, eachof which is optionally substituted with 1, 2, or 3 of R^(D). Each R^(D)is —Z^(D)R₉, wherein each Z^(D) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(D) are optionally and independently replaced by —CO—,—CS—, —CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—,—NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—,—SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—. Each R₉is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃. EachR^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In some embodiments, each R^(D) is independently —Z^(D)R₉; wherein eachZ^(D) can independently be a bond or an optionally substituted branchedor straight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —O—, —NHC(O)—,—C(O)NR^(E)—, —SO₂—, —NHSO₂—, —NHC(O)—, —NR^(E)SO₂—, —SO₂NH—,—SO₂NR^(E)—, —NH—, or —C(O)O—. In some embodiments, one carbon unit ofZ^(D) is replaced by —O—. Or, by —NHC(O)—. Or, by —C(O)NR^(E)—. Or, by—SO₂—. Or, by —NHSO₂—. Or, by —NHC(O)—. Or, by —SO—. Or, by —NR^(E)SO₂—.Or, by —SO₂NH—. Or, by —SO₂NR^(E)—. Or, by —NH—. Or, by —C(O)O—.

In some embodiments, R₉ is hydrogen. In some embodiments, R₉ isindependently an optionally substituted aliphatic. In some embodiments,R₉ is an optionally substituted cycloaliphatic. Or, is an optionallysubstituted heterocycloaliphatic. Or, is an optionally substituted aryl.Or, is an optionally substituted heteroaryl. Or, halo.

In some embodiments, one R₁ is aryl or heteroaryl, each optionallysubstituted with 1, 2, or 3 of R^(D), wherein R^(D) is defined above.

In several embodiments, one R₁ is carboxy [e.g., hydroxycarbonyl oralkoxycarbonyl]. Or, one R₁ is amido [e.g., aminocarbonyl]. Or, one R₁is amino Or, is halo. Or, is cyano. Or, hydroxyl.

In some embodiments, one R₁ that is attached to 1-position of theisoquinoline ring is aryl or heteroaryl, each optionally substitutedwith 1, 2, or 3 of R^(D), wherein R^(D) is defined above. In someembodiments, the one R₁ attached to 1-position of the isoquinoline ringis phenyl optionally substituted with 1, 2, or 3 of R^(D), wherein R^(D)is defined above. In some embodiments, the one R₁ attached to the1-position of the isoquinoline ring is heteroaryl optionally substitutedwith 1, 2, or 3 of R^(D). In several embodiments, the one R₁ attached tothe 1-position of the isoquinoline ring is 5 or 6 membered heteroarylhaving 1, 2, or 3 heteroatom independently selected from the groupconsisting of oxygen, nitrogen and sulfur. In other embodiments, the 5or 6 membered heteroaryl is substituted with 1 R^(D).

In some embodiments, one R₁ attached to the 1-position of theisoquinoline ring is a phenyl substituted with 1 R^(D). In someembodiments, one R₁ attached to the 1-position of the isoquinoline ringis a phenyl substituted with 2 R^(D). In some embodiments, one R₁attached to the 1-position of the isoquinoline ring is a phenylsubstituted with 3 R^(D).

In several embodiments, R₁ is:

wherein

W₁ is —C(O)—, —SO₂—, or —CH₂—;

D is H, hydroxyl, or an optionally substituted group selected fromaliphatic, cycloaliphatic, alkoxy, and amino; and

R^(D) is defined above.

In several embodiments, W₁ is —C(O)—. Or, W₁ is —SO₂—. Or, W₁ is —CH₂—.

In several embodiments, D is OH. Or, D is an optionally substituted C₁₋₆saliphatic or an optionally substituted C₃-C₈ cycloaliphatic. Or, D isan optionally substituted straight chain or branched alkoxy. Or, D is anoptionally substituted amino.

In several examples, D is

wherein each of A and B is independently H, an optionally substitutedC₁₋₆ aliphatic, an optionally substituted C₃-C₈ cycloaliphatic, or

A and B, taken together, form an optionally substituted 3-7 memberedheterocycloaliphatic ring;

m is an integer from 1 to 6 inclusive; and

p is 2 or 3.

In several embodiments, A is H and B is an optionally substituted C₁₋₆aliphatic. In several embodiments, B is substituted with 1, 2, or 3substituents. Or, both, A and B, are H. In several embodiments, n and pare 2 and A is an optionally substituted C₁₋₆ aliphatic. In severalembodiments, n is 2, p is 3, and A is an optionally substituted C₁₋₆aliphatic. Exemplary substituents include oxo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, dialkyamino, or an optionallysubstituted group selected from cycloaliphatic, heterocycloaliphatic,aryl, and heteroaryl.

In several embodiments, A is H and B is an optionally substituted C₁₋₆aliphatic. Or, both, A and B, are H. Exemplary substituents include oxo,alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, and an optionallysubstituted heterocycloaliphatic.

In several embodiments, B is C₁₋₆ alkyl, optionally substituted withoxo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, or an optionallysubstituted group selected from cycloaliphatic, heterocycloaliphatic,aryl, and heteroaryl. In several embodiments, B is substituted with oxo,C₁₋₆ alkyl, hydroxy, hydroxy-(C₁₋₆)alkyl, (C₁₋₆)alkoxy,(C₁₋₆)alkoxy(C₁₋₆)alkyl, C₃₋₈ cycloaliphatic, 3-8 memberedheterocycloaliphatic, phenyl, and 5-10 membered heteroaryl. In oneexample, B is C₁₋₆ alkyl substituted with optionally substituted phenyl.

In several embodiments, A and B, taken together, form an optionallysubstituted 3-7 membered heterocycloaliphatic ring. In several examples,the heterocycloaliphatic ring is optionally substituted with 1, 2, or 3substituents. Exemplary such rings include optionally substitutedpyrrolidinyl, piperidinyl, morpholinyl, and piperazinyl. Exemplarysubstituents on such rings include halo, oxo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, acyl (e.g., alkylcarbonyl), amino,amido, and carboxy. In some embodiments, the substituent is halo, oxo,alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, amido, orcarboxy.

In several embodiments, R^(D) is hydrogen, halo, or an optionallysubstituted group selected from aliphatic, cycloaliphatic, amino,hydroxy, alkoxy, carboxy, amido, carbonyl, cyano, aryl, or heteroaryl.In several examples, R^(D) is hydrogen, halo, an optionally substitutedC₁₋₆ aliphatic, or an optionally substituted alkoxy. In severalexamples, R^(D) is hydrogen, F, Cl, an optionally substituted C₁₋₆alkyl, or an optionally substituted —O(C₁₋₆ alkyl). Examples of R^(D)include hydrogen, F, Cl, methyl, ethyl, i-propyl, t-butyl, —OMe, —OEt,i-propoxy, t-butoxy, CF₃, or —OCF₃. In some examples, R^(D) is hydrogen,F, methyl, methoxy, CF₃, or —OCF₃. R^(D) can be hydrogen. R^(D) can beF. R^(D) can be methyl. R^(D) can be methoxy.

In several embodiments, R₁ is:

wherein, independently for each occurrence:

G is —O—, —CHR₉—, or —NR₉—;

X is O or H,H; and

R₉ is defined above.

In several embodiments, G is —O—. In several embodiments, G is —CHR₉—.In several embodiments, G is —NR₉—. In several embodiments, X(alpha) isO. In several embodiments, X(alpha) is H,H. In several embodiments,X(beta) is O. In several embodiments, X(beta) is H,H. In severalembodiments R₉ is aliphatic. In several embodiments, R₉ is aryl. Inseveral embodiments, R₉ is H.

In several embodiments, G is —O— and both X are H,H. In severalembodiments, G is —CHR₉— and R₉ is aryl. In several embodiments, G is—NR₉— and R₉ is aliphatic. In several embodiments, G is —NR₉— and R₉ isaryl. In several examples, G is —NR₉— and R₉ is H. In severalembodiments, G is —CHR₉—, R₉ is aryl, and both X are H,H. In severalembodiments, G is —NR₉—, R₉ is aliphatic, and both X are H,H. In severalembodiments, G is —NR₉—, R₉ is aryl, and X(beta) is O. In severalembodiments, G is —NR₉—, R₉ is H, and X(beta) is O.

In several embodiments, R₉ is methyl. In several embodiments, R₉ isphenyl. In several embodiments, G is —NR₉—, R₉ is methyl, and both X areH,H. In several embodiments, G is —NR₉—, R₉ is phenyl, X(alpha) is H,H,and X(beta) is O. In several embodiments, G is —CHR₉—, R₉ is phenyl, andboth X are H,H. In several embodiments, G is —NR₉—, R₉ is H, X(alpha) isH,H, and X(beta) is O.

In several embodiments, R₁ is:

wherein, independently for each occurrence:

Y is CH or N providing that at least one Y is N;

R₁ is defined above; and

m is an integer from 0 to 4, inclusive.

In several embodiments, the ortho Y is N. In several embodiments, themeta Y is N. In several embodiments, the para Y is N. In severalembodiments, R₁ is alkoxy, amino, hydroxy, or aliphatic. In severalembodiments, m is 0. In several embodiments, m is 1. In severalembodiments, m is 2. In several embodiments, m is 3. In severalembodiments, m is 4. In several embodiments, the ortho Y is N and themeta and para Y are CH. In several embodiments, the meta Y is N and theortho and para Y are CH. In several embodiments, the para Y is N and theortho and meta Y are CH. In several embodiments, R₁ is alkoxy. Inseveral embodiments, R₁ is methoxy. In several embodiments, the meta Yis N and the ortho and para Y are CH; R₁ is alkoxy, and m is 1. Inseveral embodiments, the meta Y is N and the ortho and para Y are CH; R₁is methoxy, and m is 1. In several embodiments, the meta Y is N and theortho and para Y are CH; R₁ is methoxy and in the para position, and mis 1.

In several embodiments, R₁ is:

wherein:

W₁ is —C(O)—, —SO₂—, or —CH₂—;

each of A and B is independently H, an optionally substituted C₁₋₆aliphatic, an optionally substituted C₃-C₈ cycloaliphatic; or

A and B, taken together, form an optionally substituted 3-7 memberedheterocycloaliphatic ring.

In some embodiments, one R₁ that is attached to the 1-position of theisoquinoline ring is cycloaliphatic or heterocycloaliphatic, eachoptionally substituted with 1, 2, or 3 of R^(D); wherein R^(D) is—Z^(D)R₉; wherein each Z^(D) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(D) are optionally and independently replaced by —CO—,—CS—, —CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—,—NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—,—SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—; each R₉is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃; andeach R^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In several examples, one R₁ that is attached to the 1-position of theisoquinoline ring is an optionally substituted C₃-C₈ cycloaliphatic.

In some embodiments, one R₁ that is attached to the 1-position of theisoquinoline ring is an optionally substituted C₃-C₈ cycloalkyl or anoptionally substituted C₃-C₈ cycloalkenyl.

In several embodiments, one R₁ that is attached to the 1-position of theisoquinoline ring is C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl. Examples ofcycloalkyl and cycloalkenyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, andcycloheptenyl.

In some embodiments, one R₁ that is attached to the 1-position of theisoquinoline ring is an optionally substituted C₃-C₈ cycloaliphatic oran optionally substituted C₃-C₈ heterocycloaliphatic. In someembodiments, R₁ is an optionally substituted piperidine ring. In someembodiments, R₁ is an optionally substituted morpholine ring. In someembodiments, R₁ is an optionally substituted piperizine ring. In someembodiments, R₁ is an optionally substituted tetrahydro-2-pyrazinonering.

In some embodiments, R₁ is:

In several examples, R₁ is one selected from:

B. Substituent R₂

Each R₂ can be hydrogen. Each R₂ can be an optionally substituted groupselected from C₁₋₆ aliphatic, C₃₋₆ cycloaliphatic, phenyl, andheteroaryl.

In several embodiments, R₂ is a C₁₋₆ aliphatic optionally substitutedwith 1, 2, or 3 halo, C₁₋₂ aliphatic, or alkoxy. In several examples, R₂can be substituted methyl, ethyl, propyl, or butyl. In several examples,R₂ can be methyl, ethyl, propyl, or butyl.

In several embodiments, R₂ is hydrogen.

C. Substituents R₃ and R′₃

Each R₃ and R′₃ together with the carbon atom to which they are attachedform a C₃₋₇ cycloaliphatic or a heterocycloaliphatic, each of which isoptionally substituted with 1, 2, or 3 substituents.

In several embodiments, R₃ and R′₃ together with the carbon atom towhich they are attached form a C₃₋₇ cycloaliphatic or a C₃₋₇heterocycloaliphatic, each of which is optionally substituted with 1, 2,or 3 of —Z^(B)R₇, wherein each Z^(B) is independently a bond, or anoptionally substituted branched or straight C₁₋₄ aliphatic chain whereinup to two carbon units of Z^(B) are optionally and independentlyreplaced by —CO—, —CS—, —CONR^(B)—, —CONR^(B)NR^(B)—, —CO₂—, —OCO—,—NR^(B)CO₂—, —O—, —NR^(B)CONR^(B)—, —OCONR^(B)—, —NR^(B)NR^(B)—,—NR^(B)CO—, —S—, —SO—, —SO₂—, —NR^(B)—, —SO₂NR^(B)—, —NR^(B)SO₂—, or—NR^(B)SO₂NR^(B)—; each R₇ is independently R^(B), halo, —OH, —NH₂,—NO₂, —CN, —CF₃, or —OCF₃; and each R^(B) is independently hydrogen, anoptionally substituted C₁₋₈ aliphatic group, an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic, anoptionally substituted aryl, or an optionally substituted heteroaryl.

In several embodiments, R₃ and R′₃ together with the carbon atom towhich they are attached form a 3, 4, 5, or 6 membered cycloaliphaticthat is optionally substituted with 1, 2, or 3 substituents. In severalexamples, R₃, R′₃, and the carbon atom to which they are attached forman optionally substituted cyclopropyl group. In several alternativeexamples, R₃, R′₃, and the carbon atom to which they are attached forman optionally substituted cyclobutyl group. In several other examples,R₃, R′₃, and the carbon atom to which they are attached form anoptionally substituted cyclopentyl group. In other examples, R₃, R′₃,and the carbon atom to which they are attached form an optionallysubstituted cyclohexyl group. In more examples, R₃ and R′₃ together withthe carbon atom to which they are attached form an unsubstitutedcyclopropyl.

In several embodiments, R₃ and R′₃ together with the carbon atom towhich they are attached form a 5, 6, or 7 membered optionallysubstituted heterocycloaliphatic. In other examples, R₃, R′₃, and thecarbon atom to which they are attached form an optionally substitutedtetrahydropyranyl group.

In some embodiments, R₃ and R′₃ together with the carbon atom to whichthey are attached form an unsubstituted C₃₋₇ cycloaliphatic or anunsubstituted heterocycloaliphatic. In several examples, R₃ and R′₃together with the carbon atom to which they are attached form anunsubstituted cyclopropyl, an unsubstituted cyclopentyl, or anunsubstituted cyclohexyl.

D. Substituent R₄

Each R₄ is independently an optionally substituted aryl or an optionallysubstituted heteroaryl.

In several embodiments, R₄ is an aryl having 6 to 10 members (e.g., 7 to10 members) optionally substituted with 1, 2, or 3 substituents.Examples of R₄ include optionally substituted benzene, naphthalene, orindene. Or, examples of R₄ can be optionally substituted phenyl,optionally substituted naphthyl, or optionally substituted indenyl.

In several embodiments, R₄ is an optionally substituted heteroaryl.Examples of R₄ include monocyclic and bicyclic heteroaryl, such abenzofused ring system in which the phenyl is fused with one or two 4-8membered heterocycloaliphatic groups.

In some embodiments, R₄ is an aryl or heteroaryl, each optionallysubstituted with 1, 2, or 3 of —Z^(C)R₈. In some embodiments, R₄ is anaryl optionally substituted with 1, 2, or 3 of —Z^(C)R₈. In someembodiments, R₄ is phenyl optionally substituted with 1, 2, or 3 of—Z^(C)R₈. Or, R₄ is a heteroaryl optionally substituted with 1, 2, or 3of —Z^(C)R₈. Each Z^(C) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(C) are optionally and independently replaced by —CO—,—CS—, —CONR^(C)—, —CONR^(C)NR^(C)—, —CO₂—, —OCO—, —NR^(C)CO₂—, —O—,—NR^(C)CONR^(C)—, —OCONR^(C)—, —NR^(C)NR^(C)—, —NR^(C)CO—, —S—, —SO—,—SO₂—, —NR^(C)—, —SO₂NR^(C)—, —NR^(C)SO₂—, or —NR^(C)SO₂NR^(C)—. Each R₈is independently R^(C), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃. EachR^(C) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In some embodiments, two occurrences of —Z^(C)R₈, taken together withcarbons to which they are attached, form a 4-8 membered saturated,partially saturated, or aromatic ring with up to 3 ring atomsindependently selected from the group consisting of O, NH, NR^(C), andS; wherein R^(C) is defined herein.

In several embodiments, R₄ is one selected from

E. Exemplary Compound Families

In several embodiments, R₁ is an optionally substituted cyclic groupthat is attached to the core structure at the 1 position of theisoquinoline ring.

In several examples, R₁ is an optionally substituted aryl that isattached to the 1 position of the isoquinoline ring.

In more examples, R₁ is an optionally substituted heteroaryl that isattached to the 1 position of the isoquinoline ring.

In other embodiments, R₁ is an optionally substituted cycloaliphatic oran optionally substituted heterocycloaliphatic that is attached to theisoquinoline ring at the 1 position.

Accordingly, another aspect of the present invention provides compoundsof formula (II):

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R′₃,and R₄ are defined in formula I.

In some embodiments, each R₁ is aryl or heteroaryl optionallysubstituted with 1, 2, or 3 of R^(D), wherein R^(D) is —Z^(D)R₉, whereineach Z^(D) is independently a bond or an optionally substituted branchedor straight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —CO—, —CS—, —CONR^(E)—,—CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—, —NR^(E)CONR^(E)—,—OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—, —SO₂—, —NR^(E)—,—SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—; each R₉ is independentlyR^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃; each R^(E) isindependently hydrogen, an optionally substituted C₁₋₈ aliphatic group,an optionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl.

In some embodiment, each R₁ is cycloaliphatic or heterocycloaliphaticoptionally substituted with 1, 2, or 3 of R^(D); wherein R^(D) isdefined above.

In another aspect, the present invention includes compounds of formula(III):

or a pharmaceutically acceptable salt thereof, wherein R₂, R₃, R′₃, andR₄ are defined in formula I. It is understood from formula (IV) that R₁may be present at any available position on the two rings of theisoquinoline moiety as valency allows.

R^(D) is —Z^(D)R₉; wherein each Z^(D) is independently a bond or anoptionally substituted branched or straight C₁₋₆ aliphatic chain whereinup to two carbon units of Z^(D) are optionally and independentlyreplaced by —CO—, —CS—, —CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—,—NR^(E)CO₂—, —O—, —NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—,—NR^(E)CO—, —S—, —SO—, —SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or—NR^(E)SO₂NR^(E)—.

R₉ is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃.

Each R^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In several embodiments, Z^(D) is independently a bond or is anoptionally substituted branched or straight C₁₋₆ aliphatic chain whereinone carbon unit of Z^(D) is optionally replaced by —SO₂—, —CONR^(E)—,—NR^(E)SO₂—, or —SO₂NR^(E)—. For example, Z^(D) is an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein one carbonunit of Z^(D) is optionally replaced by —SO₂—. In other examples, R₉ isan optionally substituted heteroaryl or an optionally substitutedheterocycloaliphatic. In additional examples, R₉ is an optionallysubstituted heterocycloaliphatic having 1-2 nitrogen atoms, and R₉attaches directly to —SO₂— via a ring nitrogen.

In another aspect, the present invention includes compounds of formulaIV:

or a pharmaceutically acceptable salt thereof,

wherein:

T is an optionally substituted C₁₋₂ aliphatic chain, wherein each of thecarbon units is optionally and independently replaced by —CO—, —CF₂—,—CS—, —COCO—, —SO₂—, —B(OH)—, or —B(O(C₁₋₆ alkyl))-;

R₁′ is hydrogen, an optionally substituted C₁₋₆ aliphatic, an optionallysubstituted aryl, an optionally substituted heteroaryl, an optionallysubstituted 3 to 10 membered cycloaliphatic, an optionally substituted 3to 10 membered heterocycloaliphatic, carboxy, amido, amino, halo, orhydroxy;

R^(D1) is attached to carbon 3″ or 4″;

each R^(D1) and R^(D2) is —Z^(D)R₉, wherein each Z^(D) is independentlya bond or an optionally substituted branched or straight C₁₋₆ aliphaticchain wherein up to two carbon units of Z^(D) are optionally andindependently replaced by —CO—, —CS—, —CONR^(E)—, —CONR^(E)NR^(E)—,—CO₂—, —OCO—, —NR^(E)CO₂—, —O—, —NR^(E)CONR^(E)—, —OCONR^(E)—,—NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—, —SO₂—, —NR^(E)—, —SO₂NR^(E)—,—NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—;

R₉ is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃;

or R^(D1) and R^(D2), taken together with atoms to which they areattached, form a 3-8 membered saturated, partially unsaturated, oraromatic ring with up to 3 ring members independently selected from thegroup consisting of O, NH, NR^(E), and S; and

each R^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In some embodiments, T is an optionally substituted —CH₂—. In some otherembodiments, T is an optionally substituted —CH₂CH₂—. In some otherembodiments, T is —CF₂—.

In some embodiments, T is optionally substituted by —Z^(E)R₁₀; whereineach Z^(E) is independently a bond or an optionally substituted branchedor straight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(E)are optionally and independently replaced by —CO—, —CS—, —CONR^(F)—,—CONR^(F)NR^(F)—, —CO₂—, —OCO—, —NR^(F)CO₂—, —O—, —NR^(F)CONR^(F)—,—OCONR^(E)—, —NR^(F)NR^(F)—, —NR^(F)CO—, —S—, —SO—, —SO₂—, —NR^(F)—,—SO₂NR^(F)—, —NR^(F)SO₂—, or —NR^(F)SO₂NR^(F)—; R₁₀ is independentlyR^(F), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃; each R^(F) isindependently hydrogen, an optionally substituted C₁₋₈ aliphatic group,an optionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl. In one example, Z^(E) is —O—.

In some embodiments, R₁₀ can be an optionally substituted C₁₋₆ alkyl, anoptionally substituted C₂₋₆ alkenyl, an optionally substituted C₃₋₂cycloaliphatic, or an optionally substituted C₆₋₁₀ aryl. In oneembodiment, R₁₀ is methyl, ethyl, i-propyl, or t-butyl.

In some embodiments, up to two carbon units of T are optionallysubstituted by —CO—, —CS—, —B(OH)—, or —B(O(C₁₋₆ alkyl)-.

In some embodiments, T is selected from the group consisting of —CH₂—,—CH₂CH₂—, —CF₂—, —C(CH₃)₂—, —C(O)—,

—C(Phenyl)₂-, —B(OH)—, and —CH(OEt)-. In some embodiments, T is —CH₂—,—CF₂—, —C(CH₃)₂—,

or —C(Phenyl)₂-. In other embodiments, T is —CH₂H₂—, —C(O)—, —B(OH)—,and —CH(OEt)-. In several embodiments, T is —CH₂—, —CF₂—, —C(CH₃)₂—,

More preferably, T is —CH₂—, —CF₂—, or —C(CH₃)₂—. In severalembodiments, T is —CH₂—. Or, T is —CF₂—. Or, T is —C(CH₃)₂—.

In some embodiments, R₁′ is hydrogen. In some embodiments, R₁′ isindependently —Z^(A)R₅, wherein each Z^(A) is independently a bond or anoptionally substituted branched or straight C₁₋₆ aliphatic chain whereinup to two carbon units of Z^(A) are optionally and independentlyreplaced by —CO—, —CS—, —CONR^(A)—, —CONR^(A)NR^(A)—, —CO₂—, —OCO—,—NR^(A)CO₂—, —O—, NR^(A)CONR^(A)—, —OCONR^(A)—, —NR^(A)NR^(A)—,—NR^(A)CO—, —S—, —SO—, —SO₂—, —NR^(A)—, —SO₂NR^(A)—, —NR^(A)SO₂—, or—NR^(A)SO₂NR^(A)—. Each R₅ is independently R^(A), halo, —OH, —NH₂,—NO₂, —CN, —CF₃, or —OCF₃. Each R^(A) is independently an optionallysubstituted group selected from C₁₋₈ aliphatic group, a cycloaliphatic,a heterocycloaliphatic, an aryl, and a heteroaryl.

In some embodiments, R₁′ is selected from the group consisting of H,C₁₋₆ aliphatic, halo, CF₃, CHF₂, —O(C₁₋₆ aliphatic), C3-C5 cycloalkyl,or C4-C6 heterocycloalkyl containing one oxygen atom. In someembodiments, R₁′ is selected from the group consisting of H, methyl,ethyl, i-propyl, t-butyl, F. C₁, CF₃, CHF₂, —OCH₃, —OCH₂CH₃,—O-(i-propyl), or —O-(t-butyl). More preferably, R₁′ is H. Or, R₁′ ismethyl. Or, ethyl. Or, CF₃.

In some embodiments, R^(D1) is attached to carbon 3″ or 4″, and is—Z^(D)R₉, wherein each Z^(D) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(D) are optionally and independently replaced by —CO—,—CS—, —CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—,—NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—,—SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—. In yetsome embodiments, Z^(D) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein one carbonunit of Z^(D) is optionally replaced by —CO—, —SO—, —SO₂—, —COO—, —OCO—,—CONR^(E)—, —NR^(E)CO—, NR^(E)CO₂—, —O—, —NR^(E)SO₂—, or —SO₂NR^(E)—. Insome embodiments, one carbon unit of Z^(D) is optionally replaced by—CO—. Or, by —SO—. Or, by —SO₂—. Or, by —COO—. Or, by —OCO—. Or, by—CONR^(E)—. Or, by —NR^(E)CO—. Or, by —NR^(E)CO₂—. Or, by —O—. Or, by—NR^(E)SO₂—. Or, by —SO₂NR^(E)—.

In several embodiments, R₉ is hydrogen, halo, —OH, —NH₂, —CN, —CF₃,—OCF₃, or an optionally substituted group selected from the groupconsisting of C₁₋₆ aliphatic, C₃₋₈ cycloaliphatic, 3-8 memberedheterocycloaliphatic, C₆₋₁₀ aryl, and 5-10 membered heteroaryl. Inseveral examples, R₉ is hydrogen, F, Cl, —OH, —CN, —CF₃, or —OCF₃. Insome embodiments, R⁹ is C₁₋₆ aliphatic, C₃₋₈ cycloaliphatic, 3-8membered heterocycloaliphatic, C₆₋₁₀ aryl, and 5-10 membered heteroaryl,each of which is optionally substituted by 1 or 2 substituentsindependently selected from the group consisting of R^(E), oxo, halo,—OH, —NR^(E)R^(E), —OR^(E), —COOR^(E), and —CONR^(E)R^(E). In severalexamples, R₉ is optionally substituted by 1 or 2 substituentsindependently selected from the group consisting of oxo, F, Cl, methyl,ethyl, i-propyl, t-butyl, —CH₂OH, —CH₂CH₂OH, —C(O)OH, —C(O)NH₂,—CH₂O(C₁₋₆ alkyl), —CH₂CH₂O(C₁₋₆ alkyl), and —C(O)(C₁₋₆ alkyl).

In one embodiment, R₉ is hydrogen. In some embodiments, R₉ is selectedfrom the group consisting of C₁₋₆ straight or branched alkyl or C₂₋₆straight or branched alkenyl; wherein said alkyl or alkenyl isoptionally substituted by 1 or 2 substituents independently selectedfrom the group consisting of R^(E), oxo, halo, —OH, —NR^(E)R^(E),—OR^(E), —COOR^(E), and —CONR^(E)R^(E).

In other embodiments, R₉ is C₃₋₈ cycloaliphatic optionally substitutedby 1 or 2 substituents independently selected from the group consistingof R^(E), oxo, halo, —OH, —NR^(E)R^(E), —OR^(E), —COOR^(E), and—CONR^(E)R^(E). Examples of cycloaliphatic include but are not limitedto cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

In yet other embodiments, R₉ is a 3-8 membered heterocyclic with 1 or 2heteroatoms independently selected from the group consisting of O, NH,NR^(E), and S; wherein said heterocyclic is optionally substituted by 1or 2 substituents independently selected from the group R^(E), oxo,halo, —OH, —NR^(E)R^(E), —OR^(E), —COOR^(E), and —CONR^(E)R^(E). Exampleof 3-8 membered heterocyclic include but are not limited to

In yet some other embodiments, R₉ is an optionally substituted 5-8membered heteroaryl with one or two ring atom independently selectedfrom the group consisting of O, S, and NR^(E). Examples of 5-8 memberedheteroaryl include but are not limited to

In some embodiments, R^(D1) and R^(D2), taken together with carbons towhich they are attached, form an optionally substituted 4-8 memberedsaturated, partially unsaturated, or aromatic ring with 0-2 ring atomsindependently selected from the group consisting of O, NH, NR^(E), andS. Examples of R^(D1) and R^(D2), taken together with phenyl containingcarbon atoms 3″ and 4″, include but are not limited to

In some embodiments, R^(D2) is selected from the group consisting of H,R^(E), halo, —OH, —(CH₂)_(r)NR^(E)R^(E), —(CH₂)_(r)OR^(E), —SO₂—R^(E),—NR^(E)—SO₂—R^(E), —SO₂NR^(E)R^(E), —C(O)R^(E), —C(O)OR^(E),—OC(O)OR^(E), —NR^(E)C(O)OR^(E), and —C(O)NR^(E)R^(E); wherein r is 0,1, or 2. In other embodiments, R^(D2) is selected from the groupconsisting of H, C₁₋₆ aliphatic, halo, —CN, —NH₂, —NH(C₁₋₆ aliphatic),—N(C₁₋₆ aliphatic)₂, —CH₂—N(C₁₋₆ aliphatic)₂, —CH₂—NH(C₁₋₆ aliphatic),—CH₂NH₂, —OH, —O(C₁₋₆ aliphatic), —CH₂OH, —CH₂—O(C₁₋₆ aliphatic),—SO₂(C₁₋₆ aliphatic), —N(C₁₋₆ aliphatic)-SO₂(C₁₋₆ aliphatic),—NH—SO₂(C₁₋₆ aliphatic), —SO₂NH₂, —SO₂NH(C₁₋₆ aliphatic), —SO₂N(C₁₋₆aliphatic)₂, —C(O)(C₁₋₆ aliphatic), —C(O)O(C₁₋₆ aliphatic), —C(O)OH,—OC(O)O(C₁₋₆ aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)O(C₁₋₆aliphatic), —N(C₁₋₆ aliphatic)C(O)O(C₁₋₆ aliphatic), —C(O)NH₂, and—C(O)N(C₁₋₆ aliphatic)₂. In several examples, R^(D2) is selected fromthe group consisting of H, C₁₋₆ aliphatic, halo, —CN, —NH₂, —CH₂NH₂,—OH, —O(C₁₋₆ aliphatic), —CH₂OH, —SO₂(C₁₋₆ aliphatic), —NH—SO₂(C₁₋₆aliphatic), —C(O)O(C₁₋₆ aliphatic), —C(O)OH, —NHC(O)(C₁₋₆ aliphatic),—C(O)NH₂, —C(O)NH(C₁₋₆ aliphatic), and —C(O)N(C₁₋₆ aliphatic)₂. Forexamples, R^(D2) is selected from the group consisting of H, methyl,ethyl, n-propyl, i-propyl, t-butyl, F, Cl, CN, —NH₂, —CH₂NH₂, —OH,—OCH₃, —O-ethyl, —O-(i-propyl), —O-(n-propyl), —CH₂OH, —SO₂CH₃,—NH—SO₂CH₃, —C(O)OCH₃, —C(O)O CH₂CH₃, —C(O)OH, —NHC(O)CH₃, —C(O)NH₂, and—C(O)N(CH₃)₂. In one embodiment, R^(D2) is hydrogen. In anotherembodiment, R^(D2) is methyl. Or, R^(D2) is ethyl. Or, R^(D2) is F. Or,R^(D2) is Cl. Or, —OCH₃.

In another aspect, the present invention provides compounds of formulaV-A or formula V-B:

wherein T, R^(D1), R^(D2), and R₁′ are as defined above.

In one embodiment, T is —CH₂—, —CF₂—, or —C(CH₃)₂—.

In one embodiment, R₁′ is selected from the group consisting of H, C₁₋₆aliphatic, halo, CF₃, CHF₂, —O(C₁₋₆ aliphatic), C3-C5 cycloalkyl, orC4-C6 heterocycloalkyl containing one oxygen atom. Exemplary embodimentsinclude H, methyl, ethyl, i-propyl, t-butyl, F. C₁, CF₃, CHF₂, —OCH₃,—OCH₂CH₃, —O-(i-propyl), —O-(t-butyl), cyclopropyl, or oxetanyl. Morepreferably, R₁′ is H. Or, R₁′ is methyl. Or, ethyl. Or, CF₃. Or,oxetanyl.

In one embodiment, R^(D1) is Z^(D)R₉, wherein Z^(D) is selected fromCONH, NHCO, SO₂NH, SO₂N(C₁₋₆ alkyl), NHSO₂, CH₂NHSO₂, CH₂N(CH₃)SO₂,CH₂NHCO, COO, SO₂, or CO. In one embodiment, R^(D1) is Z^(D)R₉, whereinZ^(D) is selected from CONH, SO₂NH, SO₂N(C₁₋₆ alkyl), CH₂NHSO₂,CH₂N(CH₃)SO₂, CH₂NHCO, COO, SO₂, or CO.

In one embodiment, Z^(D) is COO and R₉ is H. In one embodiment, Z^(D) isCOO and R₉ is an optionally substituted straight or branched C₁₋₆aliphatic. In one embodiment, Z^(D) is COO and R₉ is an optionallysubstituted straight or branched C₁₋₆ alkyl. In one embodiment, Z^(D) isCOO and R₉ is C₁₋₆ alkyl. In one embodiment, Z^(D) is COO and R₉ ismethyl.

In one embodiment, Z^(D) is CH₂O and R₉ is H. In one embodiment, Z^(D)is CH₂O and R₉ is an optionally substituted straight or branched C₁₋₆aliphatic. In one embodiment, Z^(D) is CH₂O and R₉ is an optionallysubstituted straight or branched C₁₋₆ alkyl.

In one embodiment, Z^(D) is CONH and R₉ is H. In one embodiment, Z^(D)is CONH and R₉ is an optionally substituted straight or branched C₁₋₆aliphatic. In one embodiment, Z^(D) is CONH and R₉ is straight orbranched C₁₋₆ alkyl. In one embodiment, Z^(D) is CONH and R₉ is methyl.In one embodiment, Z^(D) is CONH and R₉ is an optionally substitutedstraight or branched C₁₋₆ alkyl. In one embodiment, In one embodiment,Z^(D) is CONH and R₉ is 2-(dimethylamino)-ethyl.

In some embodiments, Z^(D) is CO and R₉ is an optionally substitutedcycloaliphatic. In some embodiments, Z^(D) is CO and R₉ is an optionallysubstituted heterocycloaliphatic. In some embodiments, Z^(D) is CO andR₉ is —N(C₂H₄)₂NH. In some embodiments, Z^(D) is CO and R₉ is—N(C₂H₄)₂NMe. In some embodiments, Z^(D) is CO and R₉ is —N(C₂H₄)₂O.

In some embodiments, Z^(D) is CH₂NHCO and R₉ is an optionallysubstituted straight or branched C₁₋₆ aliphatic or an optionallysubstituted alkoxy. In some embodiments, Z^(D) is CH₂NHCO and R₉ isstraight or branched C₁₋₆ alkyl optionally substituted with halo, oxo,hydroxyl, or an optionally substituted group selected from aliphatic,cyclic, aryl, heteroaryl, alkoxy, amino, carboxyl, or carbonyl. In oneembodiment, Z^(D) is CH₂NHCO and R₉ is methyl. In one embodiment, Z^(D)is CH₂NHCO and R₉ is CF₃. In one embodiment, Z^(D) is CH₂NHCO and R₉ ist-butoxy.

In one embodiment, Z^(D) is SO₂NH and R₉ is H. In some embodiments,Z^(D) is SO₂NH and R₉ is an optionally substituted straight or branchedC₁₋₆ aliphatic. In some embodiments, Z^(D) is SO₂NH and R₉ is straightor branched C₁₋₆ alkyl optionally substituted with halo, oxo, hydroxyl,or an optionally substituted group selected from C₁₋₆ aliphatic, 3-8membered cyclic, C₆₋₁₀ aryl, 5-8 membered heteroaryl, alkoxy, amino,amido, carboxyl, or carbonyl. In one embodiment, Z^(D) is SO₂NH and R₉is methyl. In one embodiment, Z^(D) is SO₂NH and R₉ is ethyl. In oneembodiment, Z^(D) is SO₂NH and R₉ is i-propyl. In one embodiment, Z^(D)is SO₂NH and R₉ is t-butyl. In one embodiment, Z^(D) is SO₂NH and R₉ is3,3-dimethylbutyl. In one embodiment, Z^(D) is SO₂NH and R₉ is CH₂CH₂OH.In one embodiment, Z^(D) is SO₂NH and R₉ is CH(CH₃)CH₂OH. In oneembodiment, Z^(D) is SO₂NH and R₉ is CH₂CH(CH₃)OH. In one embodiment,Z^(D) is SO₂NH and R₉ is CH(CH₂OH)₂. In one embodiment, Z^(D) is SO₂NHand R₉ is CH₂CH(OH)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH(OH)CH₂CH₃. In one embodiment, Z^(D) is SO₂NH and R₉ isC(CH₃)₂CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH(CH₂CH₃)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH₂OCH₂CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isC(CH₃)(CH₂OH)₂. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH(OH)CH₂C(O)OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH₂N(CH₃)₂. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH₂NHC(O)CH₃. In one embodiment, Z^(D) is SO₂NH and R₉ isCH(CH(CH₃)₂)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH(CH₂CH₂CH₃)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ is1-tetrahydrofuryl-methyl. In one embodiment, Z^(D) is SO₂NH and R₉ isfurylmethyl. In one embodiment, Z^(D) is SO₂NH and R₉ is(5-methylfuryl)-methyl. In one embodiment, Z^(D) is SO₂NH and R₉ is2-pyrrolidinylethyl. In one embodiment, Z^(D) is SO₂NH and R₉ is2-(1-methylpyrrolidinyl)-ethyl. In one embodiment, Z^(D) is SO₂NH and R₉is 2-(4-morpholinyl)-ethyl. In one embodiment, Z^(D) is SO₂NH and R₉ is3-(4-morpholinyl)-propyl.

In one embodiment, Z^(D) is SO₂NH and R₉ is C(CH₂CH₃)(CH₂OH)₂. In oneembodiment, Z^(D) is SO₂NH and R₉ is 2-(1H-imidazol-4-yl)ethyl. In oneembodiment, Z^(D) is SO₂NH and R₉ is 3-(1H-imidazol-1-yl)-propyl. In oneembodiment, Z^(D) is SO₂NH and R₉ is 2-(2-pyridinyl)-ethyl.

In some embodiment, Z^(D) is SO₂NH and R₉ is an optionally substitutedC₁₋₆ cycloaliphatic. In several examples, Z^(D) is SO₂NH and R₉ is anoptionally substituted C₁₋₆ cycloalkyl. In several examples, Z^(D) isSO₂NH and R₉ is C₁₋₆ cycloalkyl. In one embodiment, Z^(D) is SO₂NH andR₉ is cyclobutyl. In one embodiment, Z^(D) is SO₂NH and R₉ iscyclopentyl. In one embodiment, Z^(D) is SO₂NH and R₉ is cyclohexyl.

In some embodiments, Z^(D) is SO₂N(C₁₋₆ alkyl) and R₉ is an optionallysubstituted straight or branched C₁₋₆ aliphatic or an optionallysubstituted cycloaliphatic. In some embodiments, Z^(D) is SO₂N(C₁₋₆alkyl) and R₉ is an optionally substituted straight or branched C₁₋₆aliphatic. In some embodiments, Z^(D) is SO₂N(C₁₋₆ alkyl) and R₉ is anoptionally substituted straight or branched C₁₋₆ alkyl or an optionallysubstituted straight or branched C₁₋₆ alkenyl. In one embodiments, Z^(D)is SO₂N(CH₃) and R₉ is methyl. In one embodiments, Z^(D) is SO₂N(CH₃)and R₉ is n-propyl. In one embodiments, Z^(D) is SO₂N(CH₃) and R₉ isn-butyl. In one embodiments, Z^(D) is SO₂N(CH₃) and R₉ is cyclohexyl. Inone embodiments, Z^(D) is SO₂N(CH₃) and R₉ is allyl. In one embodiments,Z^(D) is SO₂N(CH₃) and R₉ is CH₂CH₂OH. In one embodiments, Z^(D) isSO₂N(CH₃) and R₉ is CH₂CH(OH)CH₂OH. In one embodiments, Z^(D) isSO₂N(CH₂CH₂CH₃) and R₉ is cyclopropylmethyl.

In one embodiment, Z^(D) is CH₂NHSO₂ and R₉ is methyl. In oneembodiment, Z^(D) is CH₂N(CH₃)SO₂ and R₉ is methyl.

In some embodiments, Z^(D) is SO₂ and R₉ is an optionally substitutedC₁₋₆ straight or branched aliphatic or an optionally substituted 3-8membered heterocyclic, having 1, 2, or 3 ring members selected from thegroup consisting of nitrogen, oxygen, sulfur, SO, or SO₂. In someembodiments, Z^(D) is SO₂ and R₉ is straight or branched C₁₋₆ alkyl or3-8 membered heterocycloaliphatic each of which is optionallysubstituted with 1, 2, or 3 of oxo, halo, hydroxyl, or an optionallysubstituted group selected from C₁₋₆ aliphatic, carbonyl, amino, andcarboxy. In one embodiment, Z^(D) is SO₂ and R₉ is methyl. In someembodiments, Z^(D) is SO₂ and examples of R₉ include

In some embodiments, R^(D2) is H, hydroxyl, halo, C₁₋₆ alkyl, C₁₋₆alkoxy, C₃₋₆ cycloalkyl, or NH₂. In several examples, R^(D2) is H, halo,C₁₋₄ alkyl, or C₁₋₄ alkoxy. Examples of R^(D2) include H, F, Cl, methyl,ethyl, and methoxy.

In another aspect, the present invention provides compounds of formulaVI:

wherein G is —O—, —CHR₉—, or —NR₉—;

X is O or H,H;

R₁₀ and R₁₁ are independently H, an optionally substituted C₁₋₆aliphatic, an optionally substituted aryl, an optionally substitutedheteroaryl, an optionally substituted C₃₋₁₀ cycloaliphatic, anoptionally substituted 3 to 10 membered heterocycloaliphatic, carboxy[e.g., hydroxycarbonyl or alkoxycarbonyl], amido [e.g., aminocarbonyl],amino, halo, alkoxy, cyano, or hydroxy; or R₁₀ and R₁₁ taken togetherform

and

R₉, T, and R₁′ are defined above.

In some embodiments, R₁′ is selected from the group consisting of H,C₁₋₆ aliphatic, halo, CF₃, CHF₂, —O(C₁₋₆ aliphatic), C3-C5 cycloalkyl,or C4-C6 heterocycloalkyl containing one oxygen atom. Exemplaryembodiments include H, methyl, ethyl, i-propyl, t-butyl, F. C₁, CF₃,CHF₂, —OCH₃, —OCH₂CH₃, —O-(i-propyl), —O-(t-butyl), cyclopropyl, oroxetanyl. More preferably, R₁′ is H. Or, R₁′ is methyl. Or, ethyl. Or,CF₃. Or, oxetanyl.

In some embodiments, G is —O—. In some embodiments, G is —CHR₉—. In someembodiments, G is —NR₉—. In some embodiments, X is O. In someembodiments, X is H,H. In some embodiments, R₉ is aliphatic. In someembodiments, R₉ is aryl. In some embodiments, R₉ is H. In someembodiments, R₁₁ is hydroxy, amino, or alkoxy. In some embodiments R₁₀is H. In some embodiments, R₁₀ and R₁₁ taken together form

In some embodiments, G is —O— and X is H,H. In some embodiments, G is—CHR₉— and R₉ is aryl. In some embodiments, G is —NR₉— and R₉ isaliphatic. In some embodiments, G is —NR₉— and R₉ is aryl. In someembodiments, G is —NR₉— and R₉ is H. In some embodiments, G is —CHR₉—,R₉ is aryl, and X is H,H. In some embodiments, G is —NR₉—, R₉ isaliphatic, and X is H,H. In some embodiments, G is —NR₉—, R₉ is aryl,and X is O. In some embodiments, G is —NR₉—, R₉ is H, and X is O. Insome embodiments, G is —CHR₉—, R₉ is aryl, X is H,H, R₁₁ is alkoxy, andR₁₀ is H. In some embodiments, G is —NR₉—, R₉ is aliphatic, X is H,H,R₁₁ is alkoxy, and R₁₀ is H. In some embodiments, G is —NR₉—, R₉ isaryl, X is O, R₁₁ is alkoxy, and R₁₀ is H. In some embodiments, G is—NR₉—, R₉ is H, X is O, R₁₁ is alkoxy, and R₁₀ is H.

In some embodiments, R₉ is methyl. In some embodiments, R₉ is phenyl. Insome embodiments, G is —NR₉—, R₉ is methyl, and X is H,H. In someembodiments, G is —NR₉—, R₉ is phenyl, and X is O. In some embodiments,G is —CHR₉—, R₉ is phenyl, and X is H,H. In some embodiments, G is—NR₉—, R₉ is H, and X is O. In some embodiments, G is —NR₉—, R₉ ismethyl, X is H,H, R₁₁ is methoxy, and R₁₀ is H. In some embodiments, Gis —NR₉—, R₉ is phenyl, X is O, R₁₁ is methoxy, and R₁₀ is H. In someembodiments, G is —CHR₉—, R₉ is phenyl, X is H,H, R₁₁ is methoxy, andR₁₀ is H. In some embodiments, G is —NR₉—, R₉ is H, X is O, R₁₁ ismethoxy, and R₁₀ is H.

In some embodiments, G is —CHR₉—, R₉ is aryl, X is H,H, and R₁₀ and R₁₁taken together form

In some embodiments, G is —NR₉—, R₉ is aliphatic, X is H,H, and R₁₀ andR₁₁ taken together form

In some embodiments, G is —NR₉—, R₉ is aryl, X is O, and R₁₀ and R₁₁taken together form

In some embodiments, G is —N₉—, R₉ is H, X is O, and R₁₀ and R₁₁ takentogether form

In some embodiments, G is —NR₉—, R₉ is methyl, X is H,H, and R₁₀ and R₁₁taken together form

wherein T is —CH₂—, —CF₂—, or —C(CH₃)₂—. In some embodiments, G is—NR₉—, R₉ is phenyl, X is O, and R₁₀ and R₁₁ taken together form

wherein T is —CH₂—, —CF₂—, or —C(CH₃)₂—. In some embodiments, G is—CHR₉—, R₉ is phenyl, X is H,H, and R₁₀ and R₁₁ taken together form

wherein T is —CH₂—, —CF₂—, or —C(CH₃)₂—. In some embodiments, G is—NR₉—, R₉ is H, X is O, and R₁₀ and R₁₁ taken together form

wherein T is —CH₂—, —CF₂—, or —C(CH₃)₂—.

In another aspect, the present invention provides compounds of formulaVII:

wherein Y is CH or N providing that at least one Y is N; m is an integerfrom 0 to 4 inclusive, and T, R₁, R₁′ are defined above.

In some embodiments, T is —CH₂—, —CF₂—, or —C(CH₃)₂—.

In some embodiments, R₁′ is selected from the group consisting of H,C₁₋₆ aliphatic, halo, CF₃, CHF₂, —O(C₁₋₆ aliphatic), C3-C5 cycloalkyl,or C4-C6 heterocycloalkyl containing one oxygen atom. Exemplaryembodiments include H, methyl, ethyl, i-propyl, t-butyl, F. C₁, CF₃,CHF₂, —OCH₃, —OCH₂CH₃, —O-(i-propyl), —O-(t-butyl), cyclopropyl, oroxetanyl. More preferably, R₁′ is H. Or, R₁′ is methyl. Or, ethyl. Or,CF₃. Or, oxetanyl.

In some embodiments, the ortho Y is N. In some embodiments, the meta Yis N. In some embodiments, the para Y is N. In some embodiments, R₁ isalkoxy, amino, hydroxy, or aliphatic. In some embodiments, m is 0. Insome embodiments, m is 1. In some embodiments, m is 2. In someembodiments, m is 3. In some embodiments, m is 4. In some embodiments,the ortho Y is N and the meta and para Y are CH. In some embodiments,the meta Y is N and the ortho and para Y are CH. In some embodiments,the para Y is N and the ortho and meta Y are CH. In some embodiments, R₁is alkoxy. In some embodiments, R₁ is methoxy. In some embodiments, themeta Y is N and the ortho and para Y are CH; R₁ is alkoxy, and m is 1.In some embodiments, the meta Y is N and the ortho and para Y are CH; R₁is methoxy, and m is 1. In some embodiments, the meta Y is N and theortho and para Y are CH; R₁ is methoxy and in the para position, and mis 1.

Exemplary compounds of the present invention include, but are notlimited to, those illustrated in Table 1 below.

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

Synthetic Schemes

Compounds of the invention may be prepared by known methods or asillustrated in the examples. In one instance wherein R₁ is aryl orheteroaryl, the compounds of the invention may be prepared asillustrated in Scheme I.

PG=protecting group; a) PG=COR: RCOCl, Et₃N; b) H₂O₂/AcOH, CH₃ReO₃/H₂O₂,or mCPBA; c) POCl₃, Et₃N; d) acid or basic de-protection conditions suchas 6N HCl or 1N NaOH.

Referring to Scheme I, a nitrile of formula i is alkylated (step a) witha dihalo-aliphatic in the presence of a base such as, for example, 50%sodium hydroxide and, optionally, a phase transfer reagent such as, forexample, benzyltriethylammonium chloride (BTEAC), to produce thecorresponding alkylated nitrile (not shown) which on hydrolysis producesthe acid ii. Compounds of formula II are converted to the acid chlorideiii with a suitable reagent such as, for example, thionyl chloride/DMF.Reaction of the acid chloride iii with an aminopyridine, wherein X is ahalo, of formula iv (step c) produces the amide of formula v. Reactionof the amide v with an optionally substituted boronic acid derivative(step d) in the presence of a catalyst such as, for example, palladiumacetate or dichloro-[1,1-bis(diphenylphosphino) ferrocene]palladium(II)(Pd(dppf)Cl₂), provides compounds of the invention wherein R₁ is aryl,heteroaryl, or cycloalkenyl. The boronic acid derivatives vi arecommercially available or may be prepared by known methods such asreaction of an aryl bromide with a diborane ester in the presence of acoupling reagent such as, for example, palladium acetate as described inthe examples.

In another instance where one R₁ is aryl and another R₁ is an aliphatic,alkoxy, cycloaliphatic, or heterocycloaliphatic, compounds of theinvention can be prepared as described in steps a, b, and c of Scheme Iusing an appropriately substituted isoquinoline such as

where X is halo and Q is C₁₋₆ aliphatic, aryl, heteroaryl, or 3 to 10membered cycloaliphatic or heterocycloaliphatic as a substitute for theaminopyridine of formula iv.

Formulations, Administrations, and Uses

Pharmaceutically Acceptable Compositions

Accordingly, in another aspect of the present invention,pharmaceutically acceptable compositions are provided, wherein thesecompositions comprise any of the compounds as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle. In certain embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need is capable of providing,directly or indirectly, a compound as otherwise described herein, or ametabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitory active metabolite or residuethereof.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington: TheScience and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, the contents of each of which isincorporated by reference herein, disclose various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by ABC transporteractivity. In certain embodiments, the present invention provides amethod of treating a condition, disease, or disorder implicated by adeficiency of ABC transporter activity, the method comprisingadministering a composition comprising a compound of formulae (I, II,III, IV, V-A, V-B, VI, and VII or sub-classes thereof) to a subject,preferably a mammal, in need thereof.

In certain preferred embodiments, the present invention provides amethod of treating cystic fibrosis, asthma, smoke induced COPD, chronicbronchitis, rhinosinusitis, constipation, pancreatitis, pancreaticinsufficiency, male infertility caused by congenital bilateral absenceof the vas deferens (CBAVD), mild pulmonary disease, idiopathicpancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liverdisease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, comprising the step of administering to said mammal aneffective amount of a composition comprising a compound of formulae (I,II, III, IV, V-A, V-B, VI, and VII or sub-classes thereof), or apreferred embodiment thereof as set forth above.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of formulae (I, II, III, IV, V-A, V-B, VI, and VIIor sub-classes thereof), or a preferred embodiment thereof as set forthabove.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of cystic fibrosis,asthma, smoke induced COPD, chronic bronchitis, rhinosinusitis,constipation, pancreatitis, pancreatic insufficiency, male infertilitycaused by congenital bilateral absence of the vas deferens (CBAVD), mildpulmonary disease, idiopathic pancreatitis, allergic bronchopulmonaryaspergillosis (ABPA), liver disease, hereditary emphysema, hereditaryhemochromatosis, coagulation-fibrinolysis deficiencies, such as proteinC deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetesinsipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, progressive supranuclear plasy, Pick's disease, severalpolyglutamine neurological disorders such as Huntington, spinocerebullarataxia type I, spinal and bulbar muscular atrophy, dentatorubalpallidoluysian, and myotonic dystrophy, as well as spongiformencephalopathies, such as hereditary Creutzfeldt-Jakob disease (due toprion protein processing defect), Fabry disease, Straussler-Scheinkersyndrome, COPD, dry-eye disease, or Sjogren's disease.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of cystic fibrosis, asthma, smoke induced COPD, chronicbronchitis, rhinosinusitis, constipation, pancreatitis, pancreaticinsufficiency, male infertility caused by congenital bilateral absenceof the vas deferens (CBAVD), mild pulmonary disease, idiopathicpancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liverdisease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

As described generally above, the compounds of the invention are usefulas modulators of ABC transporters. Thus, without wishing to be bound byany particular theory, the compounds and compositions are particularlyuseful for treating or lessening the severity of a disease, condition,or disorder where hyperactivity or inactivity of ABC transporters isimplicated in the disease, condition, or disorder. When hyperactivity orinactivity of an ABC transporter is implicated in a particular disease,condition, or disorder, the disease, condition, or disorder may also bereferred to as an “ABC transporter-mediated disease, condition ordisorder”. Accordingly, in another aspect, the present inventionprovides a method for treating or lessening the severity of a disease,condition, or disorder where hyperactivity or inactivity of an ABCtransporter is implicated in the disease state.

The activity of a compound utilized in this invention as a modulator ofan ABC transporter may be assayed according to methods describedgenerally in the art and in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

In one embodiment, the additional agent is selected from a mucolyticagent, a bronchodialator, an antibiotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator, or a nutritional agent.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPmodulators such as rolipram, sildenafil, milrinone, tadalafil, aminone,isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin,lactacystin, etc.

In another embodiment, the additional agent is a compound disclosed inWO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740.

In another embodiment, the additional agent is a benzo(c)quinoliziniumderivative that exhibits CFTR modulation activity or a benzopyranderivative that exhibits CFTR modulation activity.

In another embodiment, the additional agent is a compound disclosed inU.S. Pat. No. 7,202,262, U.S. Pat. No. 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502.

In another embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006002421,WO2006099256, WO2006127588, or WO2007044560.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating ABC transporteractivity in a biological sample or a patient (e.g., in vitro or invivo), which method comprises administering to the patient, orcontacting said biological sample with a compound of formula I or acomposition comprising said compound. The term “biological sample”, asused herein, includes, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof.

Modulation of ABC transporter activity in a biological sample is usefulfor a variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to, the study ofABC transporters in biological and pathological phenomena; and thecomparative evaluation of new modulators of ABC transporters.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of formulae (I, II, III, IV,V-A, V-B, VI, and VII or sub-classes thereof). In preferred embodiments,the anion channel is a chloride channel or a bicarbonate channel. Inother preferred embodiments, the anion channel is a chloride channel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional ABC transporters in amembrane of a cell, comprising the step of contacting said cell with acompound of formula (I, II, III, IV, V-A, V-B, VI, and VII orsub-classes thereof). The term “functional ABC transporter” as usedherein means an ABC transporter that is capable of transport activity.In preferred embodiments, said functional ABC transporter is CFTR.

According to another preferred embodiment, the activity of the ABCtransporter is measured by measuring the transmembrane voltagepotential. Means for measuring the voltage potential across a membranein the biological sample may employ any of the known methods in the art,such as optical membrane potential assay or other electrophysiologicalmethods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo comprising (i) a compositioncomprising a compound of formula (I, II, III, IV, V-A, V-B, VI, and VIIor sub-classes thereof) or any of the above embodiments; and (ii)instructions for a.) contacting the composition with the biologicalsample and b.) measuring activity of said ABC transporter or a fragmentthereof. In one embodiment, the kit further comprises instructions fora.) contacting an additional composition with the biological sample; b.)measuring the activity of said ABC transporter or a fragment thereof inthe presence of said additional compound, and c.) comparing the activityof the ABC transporter in the presence of the additional compound withthe density of the ABC transporter in the presence of a composition offormula (I, II, III, IV, V-A, V-B, VI, and VII or sub-classes thereof).In preferred embodiments, the kit is used to measure the density ofCFTR.

EXAMPLES Preparation:1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylic acid

Step a: 2,2-Difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester

A solution of 5-bromo-2,2-difluoro-benzo[1,3]dioxole (11.8 g, 50.0 mmol)and tetrakis(triphenylphosphine)palladium (0) [Pd(PPh₃)₄, 5.78 g, 5.00mmol] in methanol (20 mL) containing acetonitrile (30 mL) andtriethylamine (10 mL) was stirred under a carbon monoxide atmosphere (55PSI) at 75° C. (oil bath temperature) for 15 hours. The cooled reactionmixture was filtered and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography to give crude2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester (11.5 g),which was used directly in the next step.

Step b: (2,2-Difluoro-benzo[1,3]dioxol-5-yl)-methanol

Crude 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester(11.5 g) dissolved in 20 mL of anhydrous tetrahydrofuran (THF) wasslowly added to a suspension of lithium aluminum hydride (4.10 g, 106mmol) in anhydrous THF (100 mL) at 0° C. The mixture was then warmed toroom temperature. After being stirred at room temperature for 1 hour,the reaction mixture was cooled to 0° C. and treated with water (4.1 g),followed by sodium hydroxide (10% aqueous solution, 4.1 mL). Theresulting slurry was filtered and washed with THF. The combined filtratewas evaporated to dryness and the residue was purified by silica gelcolumn chromatography to give(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 76% over twosteps) as a colorless oil.

Step c: 5-Chloromethyl-2,2-difluoro-benzo[1,3]dioxole

Thionyl chloride (45 g, 38 mmol) was slowly added to a solution of(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol) indichloromethane (200 mL) at 0° C. The resulting mixture was stirredovernight at room temperature and then evaporated to dryness. Theresidue was partitioned between an aqueous solution of saturated sodiumbicarbonate (100 mL) and dichloromethane (100 mL). The separated aqueouslayer was extracted with dichloromethane (150 mL) and the organic layerwas dried over sodium sulfate, filtered, and evaporated to dryness togive crude 5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole (4.4 g) whichwas used directly in the next step.

Step d: (2,2-Difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile

A mixture of crude 5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole (4.4 g)and sodium cyanide (1.36 g, 27.8 mmol) in dimethylsulfoxide (50 mL) wasstirred at room temperature overnight. The reaction mixture was pouredinto ice and extracted with ethyl acetate (300 mL). The organic layerwas dried over sodium sulfate and evaporated to dryness to give crude(2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile (3.3 g) which was useddirectly in the next step.

Step e: 1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile

Sodium hydroxide (50% aqueous solution, 10 mL) was slowly added to amixture of crude (2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile,benzyltriethylammonium chloride (3.00 g, 15.3 mmol), and1-bromo-2-chloroethane (4.9 g, 38 mmol) at 70° C. The mixture wasstirred overnight at 70° C. before the reaction mixture was diluted withwater (30 mL) and extracted with ethyl acetate. The combined organiclayers were dried over sodium sulfate and evaporated to dryness to givecrude 1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile,which was used directly in the next step.

Step f: 1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylicacid

1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile (crudefrom the last step) was refluxed in 10% aqueous sodium hydroxide (50 mL)for 2.5 hours. The cooled reaction mixture was washed with ether (100mL) and the aqueous phase was acidified to pH 2 with 2M hydrochloricacid. The precipitated solid was filtered to give1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylic acid as awhite solid (0.15 g, 2% over four steps). ESI-MS m/z calc. 242.0. found241.6 (M+1)⁺; ¹H NMR (CDCl₃) δ 7.14-7.04 (m, 2H), 6.98-6.96 (m, 1H),1.74-1.64 (m, 2H), 1.26-1.08 (m, 2H).

Preparation:N-(1-Bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo-[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

Step a: 1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonylchloride

To 1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid(25.0 g, 103 mmol) in thionyl chloride (22.5 mL, 309 mmol) was addedN,N-dimethylformamide (200 μL). The reaction mixture was stirred at roomtemperature for 2 h. Excess thionyl chloride and N, N-dimethylformamidewere removed in vacuo to yield1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride(26.3 g, 83%)

Step b:N-(1-Bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

To a solution of 1-bromoisoquinolin-3-amine (3.00 g, 13.5 mmol) and Et₃N(3.8 mL, 27 mmol) in dichloromethane (50 mL) was added a solution of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride(4.18 g, 13.5 mmol) in dichloromethane (50 mL). The resulting reactionmixture was allowed to stir at room temperature for 18 h. The reactionmixture was then washed with 1N aqueous NaOH (2×200 mL), 1 N aqueous HCl(1×200 mL) and saturated aqueous NaHCO₃ (1×200 mL). The organics weredried over sodium sulfate and evaporated. The resulting material waspurified by silica gel chromatography eluting with 0-50% ethylacetate/hexanes to yieldN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]-dioxol-5-yl)cyclopropanecarboxamide(4.2 g, 70%). ESI-MS m/z calc. 446.0. found 447.1 (M+1)⁺. Retention time2.39 minutes.

Preparation:N-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamide

To 1-(4-methoxyphenyl)cyclopropanecarboxylic acid (4.07 g, 21.17 mmol),thionyl chloride (4.64 mL, 63.52 mmol) and DMF (64 μL) were stirred at50° C. for 3 hours, after which additional thionyl chloride (4 mL) andDMF (60 μL) were added and the mixture was stirred at 50° C. for 1additional hour. The excess thionyl chloride was evaporated underreduced pressure. The resulting acid chloride was dissolved in anhydrousDCM (20 mL) and was slowly added to a cooled suspension of (0° C.) of1-bromoisoquinolin-3-amine in DCM (50 mL) and Et₃N (14.05 mL, 100.8mmol). The reaction mixture was stirred at room temperature for 18hours. The resulting mixture was diluted with DCM and washed with water(1×30 mL), 1 N NaOH (2×30 mL), 1 N HCl (1×30 mL), saturated aqueousNaHCO₃ (1×30 mL) and brine (1×30 mL). The organic layer was dried overanhydrous Na₂SO₄ and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (0-50% ethylacetate in hexane) to yieldN-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamide(6.0 g, 75%) as a yellow solid. ESI-MS m/z calc. 396.05. found 397.3(M+1)⁺. Retention time 2.24 minutes. ¹H NMR (400.0 MHz, CDCl₃) d 8.55(s, 1H), 8.15 (d, J=8.5 Hz, 1H), 7.89 (s, 1H), 7.78 (d, J=8.2 Hz, 1H),7.69-7.65 (m, 1H), 7.56-7.52 (m, 1H), 7.46-7.42 (m, 2H), 7.01-6.98 (m,2H), 3.90 (s, 3H), 1.75 (dd, J=3.7, 6.8 Hz, 2H) and 1.21 (dd, J=3.7, 6.9Hz, 2H) ppm.

Preparation: 6-Bromoisoindolin-1-one

Step a: 5-Bromo-2-methylbenzoic acid

2-Methylbenzoic acid (40.0 g, 290 mmol) was added to a suspension of Br₂(160 mL) and iron powder (3.20 g, 57.0 mol) under N₂ atmosphere in anice bath. The mixture was allowed to warm to room temperature and wasstirred for 2 hours. The reaction mixture was poured into water and thereddish solid was collected by filtration. The solid was dried undervacuum at 50° C. The solid was dissolved in 400 mL of methanol before640 mL of 0.1N aqueous HCl was added at room temperature. The mixturewas stirred and a white solid was produced. This solid wasrecrystallized from ethanol to afford 5-bromo-2-methyl-benzoic acid(12.0 g, 19%). ¹H NMR (300M Hz, CDCl₃) δ 8.17 (d, J=2.1, 1H), 7.56 (dd,J=8.1, 2.1, 1H), 7.15 (d, J=8.1, 1H), 2.59 (s, 3H).

Step b: 5-Bromo-2-methylbenzoic acid methyl ester

To a solution of 5-bromo-2-methyl-benzoic acid (9.9 g, 46 mmol) in DMF(100 mL) was added K₂CO₃ (7.6 g, 55 mmol) and CH₃I (20 g, 140 mmol)slowly. After stirring at room temperature for 4 h, the solvent wasremoved under vacuum. The residue was partitioned between ethyl acetateand water. The organic layer was washed with brine and dried overNa₂SO₄. The solvent was removed under vacuum to afford5-bromo-2-methylbenzoic acid methyl ester (8.6 g, 82%), which was usedin next step without further purification. ¹H NMR (300 MHz, CDCl₃) δ8.04 (d, J=2.1, 1H), 7.50 (dd, J=8.1, 2.1, 1H), 7.12 (d, J=8.1, 1H),3.89 (s, 3H), 2.53 (s, 3H).

Step c: 5-Bromo-2-bromomethylbenzoic acid methyl ester

To a solution of 5-bromo-2-methylbenzoic acid methyl ester (8.4 g, 37mmol) in 100 mL CCl₄ was added N-bromosuccinimide (7.8 g, 44 mmol) andbenzoylperoxide (0.5% as catalyst). The mixture was heated at reflux for2 h and then was cooled to room temperature. The solvent was removed invacuo and the residue was purified by column chromatography on silicagel (petroleum ether) to afford 5-bromo-2-bromomethyl-benzoic acidmethyl ester (5.2 g, 46%). ¹H NMR (300 MHz, CDCl₃) δ 8.09 (s, 1H), 7.60(d, J=8.0, 1H), 7.32 (d, J=8.0, 1H), 4.89 (s, 2H), 3.94 (s, 3H).

Step d: 6-Bromoisoindolin-1-one

To a saturated solution of NH₃ in CH₃OH (50 mL) was added5-bromo-2-bromomethyl-benzoic acid methyl ester (4.8 g, 16 mmol). Thereaction mixture was stirred in a sealed tube at 40° C. overnight. Themixture was cooled to room temperature and the resultant white solid wascollected to afford 6-bromoisoindolin-1-one (2.2 g, 67%). ¹H NMR (400MHz, DMSO) δ 8.71 (s, 1H), 7.75 (d, 2H), 7.53 (s, 1H), 4.32 (s, 2H).

Preparation:6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-1-one

6-Bromoisoindolin-1-one (636 mg, 3.10 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (930 mg,3.70 mmol), and Pd(dppf)Cl₂ (125 mg, 0.150 mmol) were added to a dryflask and placed under N₂. Potassium acetate (900 mg, 9.20 mmol) wasweighed directly into the flask. The flask was then evacuated and backfilled with N₂. Anhydrous N,N-dimethylformamide (DMF) (18 mL) was addedand the reaction was heated at 80° C. overnight. The reaction mixturewas evaporated to dryness and the resulting material was purified bysilica gel chromatography eluting with 0-100% ethyl acetate in hexane toyield 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-1-one(493 mg, 62%). ESI-MS m/z calc. 259.1. found 260.1 (M+1)⁺. Retentiontime 1.24 minutes.

Preparation:N,3-Dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide

Step a: 4-Bromo-N,3-dimethylbenzenesulfonamide

To a solution of 4-bromo-3-methylbenzene-1-sulfonyl chloride (500 mg,1.86 mmol) and DIEA (0.65 mL, 3.7 mmol) in N,N-dimethylformamide (5 mL)was added methylamine as a 2.0 M solution in methanol. The reactionmixture was allowed to stir at room temperature overnight. The reactionmixture was evaporated to dryness and was dissolved in dichloromethane(3 mL). The solution was washed with 1 N HCl (2×3 mL) and a saturatedsolution of NaHCO₃ (3 mL). The organics were dried over Na₂SO₄ andevaporated to dryness to give 4-bromo-N,3-dimethylbenzenesulfonamide(340 mg).

Step b:N,3-Dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide

4-Bromo-N,3-dimethylbenzenesulfonamide (336 mg, 1.27 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (387 mg,1.50 mmol), and Pd(dppf)Cl₂ (49 mg, 0.060 mmol) were added to a dryflask and placed under N₂. Potassium acetate (382 mg, 3.90 mmol) wasweighed directly into the flask. The flask was then evacuated and backfilled with N₂. Anhydrous N,N-dimethylformamide (6 mL) was added and thereaction was heated at 80° C. in an oil bath overnight. The reactionmixture was evaporated to dryness. The residue was dissolved in ethylacetate (20 mL) and was washed with water (20 mL). The organics weredried over sodium sulfate and evaporated to dryness. The resultingmaterial was purified by silica gel chromatography eluting with 0-70%ethyl acetate in hexane to yieldN,3-Dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(126 mg, 32%). ESI-MS m/z calc. 311.2. found 312.1 (M+1)⁺. Retentiontime 1.74 minutes.

Preparation:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(3-oxoisoindolin-5-yl)isoquinolin-3-yl)cyclopropanecarboxamide

N-(1-Bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide(45 mg, 0.10 mmol) was dissolved in 1 mL of 1,2-dimethoxyethane in areaction tube. 6-(4,4,5,5-Tetramethyl-1,3-dioxolan-2-yl)isoindolin-1-one(38 mg, 0.15 mmol), 0.1 mL of an aqueous 2 M sodium carbonate solution,and tetrakis(triphenylphosphine)palladium(0) (6.0 mg, 0.0050 mmol) wereadded and the reaction mixture was heated at 120° C. for ten minutesunder microwave irradiation. The reaction mixture was evaporated todryness and the residue was purified by silica gel chromatographyeluting with 0-100% ethyl acetate/hexanes. ESI-MS m/z calc. 499.5. found500.3 (M+1)⁺. Retention time 1.93 minutes.

Preparation:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-[(hydroxymethyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamide

Step a:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-(hydroxymethyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamide

N-(1-Bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide(45 mg, 0.10 mmol), 4-(hydroxymethyl)phenylboronic acid (23 mg, 0.15mmol), and Pd(PPh₃)₄ (6 mg, 0.005 mmol) were combined in a reactiontube. DME (1 mL) and saturated Na₂CO₃ aqueous solution (100 μL) wereadded and the reaction vial was stirred under N₂ atmosphere at 80° C.overnight. The mixture was filtered and concentrated. The residue wasdissolved in DMSO and purified by reverse-phase HPLC to yield1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-(hydroxymethyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamide.ESI-MS m/z calc. 474.1. found 475.3 (M+1)⁺. Retention time 2.02 minutes.¹H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.41 (s, 1H), 8.00-7.96 (m,1H), 7.88-7.84 (m, 1H), 7.72 (t, J=7.1 Hz, 1H), 7.64-7.38 (m, 8H), 5.54(s, 1H), 4.61 (s, 2H), 1.58-1.55 (m, 2H), 1.22-1.20 (m, 2H).

Preparation:1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2-methyl-4-(N-methylsulfamoyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2-methyl-4-(N-methylsulfamoyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 2-methyl-4-(N-methylsulfamoyl)phenylboronic acid. ESI-MS m/z calc.551.1. found 552.3 (M+1)⁺. Retention time 2.18 minutes. ¹H NMR (400 MHz,DMSO-d6) δ 9.19 (s, 1H), 8.50 (s, 1H), 8.01 (d, J=8.3 Hz, 1H), 7.79 (s,1H), 7.76-7.72 (m, 2H), 7.59 (d, J=0.7 Hz, 1H), 7.54-7.37 (m, 6H), 2.48(d, J=4.9 Hz, 3H), 2.02 (s, 3H), 1.57-1.56 (m, 2H), 1.21-1.19 (m, 2H).

Preparation:4-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzamide

4-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 4-carbamoylphenylboronic acid. ESI-MS m/z calc. 487.1. found 488.3(M+1)⁺. Retention time 1.92 minutes. ¹H NMR (400 MHz, DMSO-d6) δ 9.10(s, 1H), 8.46 (s, 1H), 8.11-7.98 (m, 4H), 7.84 (d, J=8.5 Hz, 1H),7.78-7.72 (m, 1H), 7.66 (d, J=8.3 Hz, 2H), 7.61 (d, J=1.1 Hz, 1H),7.53-7.49 (m, 2H), 7.43-7.38 (m, 2H), 1.58-1.56 (m, 2H), 1.23-1.20 (m,2H).

Preparation:3-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzamide

3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 3-carbamoylphenylboronic acid. ESI-MS m/z calc. 487.1. found 488.3(M+1)⁺. Retention time 1.91 minutes. ¹H NMR (400 MHz, DMSO-d6) δ 9.14(s, 1H), 8.45 (s, 1H), 8.08-7.99 (m, 4H), 7.82 (d, J=8.4 Hz, 1H),7.76-7.72 (m, 2H), 7.64-7.60 (m, 2H), 7.53-7.38 (m, 4H), 1.58-1.55 (m,2H), 1.22-1.19 (m, 2H).

Preparation:N-(1-(1H-Indol-5-yl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

N-(1-(1H-Indol-5-yl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. ESI-MSm/z calc. 483.1. found 484.5 (M+1)⁺. Retention time 2.08 minutes. ¹H NMR(400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.94 (s, 1H), 8.37 (s, 1H),7.99-7.94 (m, 2H), 7.76 (s, 1H), 7.70 (t, J=7.4 Hz, 1H), 7.62 (s, 1H),7.54-7.42 (m, 5H), 7.31 (d, J=8.4 Hz, 1H), 6.53 (s, 1H), 1.58-1.55 (m,2H), 1.22-1.20 (m, 2H).

Preparation:N-(1-(1H-Indazol-5-yl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

N-(1-(1H-Indazol-5-yl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole. ESI-MSm/z calc. 484.1. found 485.5 (M+1)⁺. Retention time 2.01 minutes. ¹H NMR(400 MHz, DMSO-d6) δ 9.05 (d, J=9.9 Hz, 1H), 8.41 (s, 1H), 8.19 (s, 1H),7.99-7.92 (m, 3H), 7.74-7.67 (m, 2H), 7.63-7.56 (m, 2H), 7.51-7.38 (m,3H), 1.58-1.55 (m, 2H), 1.22-1.20 (m, 2H).

Preparation:1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-phenylisoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-phenylisoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand phenylboronic acid.

Preparation:1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2-methyl-1H-indol-5-yl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-phenylisoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 2-methyl-1H-indol-5-ylboronic acid.

Preparation:1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-(4-methylpiperazine-1-carbonyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-phenylisoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 4-(4-methylpiperazine-1-carbonyl)phenylboronic acid.

Preparation:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(3-sulfamoylphenyl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(3-sulfamoylphenyl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 3-sulfamoylphenylboronic acid. ESI-MS m/z calc. 523.1. found 524.3(M+1)⁺. Retention time 2.02 minutes. ¹H NMR (400 MHz, DMSO-d6) δ 9.22(s, 1H), 8.48 (s, 1H), 8.03-7.96 (m, 3H), 7.83-7.73 (m, 4H), 7.60-7.37(m, 6H), 1.58-1.55 (m, 2H), 1.22-1.19 (m, 2H).

Preparation:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-sulfamoylphenyl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-sulfamoylphenyl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 4-sulfamoylphenylboronic acid. ESI-MS m/z calc. 523.1. found 524.3(M+1)⁺. Retention time 2.03 minutes. ¹H NMR (400 MHz, DMSO-d6) δ 9.08(s, 1H), 8.47 (s, 1H), 8.02-7.97 (m, 3H), 7.83-7.73 (m, 4H), 7.61 (d,J=1.3 Hz, 1H), 7.54-7.38 (m, 5H), 1.58-1.56 (m, 2H), 1.23-1.20 (m, 2H).

Preparation:3-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoic acid

Step a: tert-Butyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoate

ToN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide(508 mg, 1.14 mmol) in 1,2-dimethoxyethane (11 mL) was added3-(tert-butoxycarbonyl)phenylboronic acid (328 mg, 1.48 mmol),tetrakis(triphenylphosphine)palladium (0) (131 mg, 0.114 mmol), and 2 MNa₂CO₃ (1.71 mL, 3.41 mmol). The mixture was heated at 80° C. overnightbefore it was diluted with ethyl acetate (10 mL) and washed with water(20 mL). The organics were dried over Na₂SO₄ and evaporated. Theresulting crude material was purified by silica gel chromatographyeluting with 0-10% ethyl acetate in hexanes to yield tert-butyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoate(603 mg, 97%). ESI-MS m/z calc. 544.6. found 545.3 (M+1)⁺. Retentiontime 2.76 minutes.

Step b:3-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoic acid

tert-Butyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoate(603 mg, 1.11 mmol) was dissolved in dichloromethane (6 mL) andtrifluoroacetic acid (3 mL). The reaction mixture was stirred at roomtemperature for 3.5 hours before it was evaporated to dryness to yield3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoic acid (499 mg, 75%) as the TFA salt. ESI-MS m/zcalc. 488.1. found 489.1 (M+1)⁺. Retention time 2.06 minutes.

Preparation:4-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoic acid

4-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoic acid was made by the procedure shown abovestarting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 4-(tert-butoxycarbonyl)phenylboronic acid.

Preparation:2-(3-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoyloxy)-N,N,N-trimethylethanaminiumchloride

Step a: 3-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoyl chloride

To3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoicacid (1.26 g, 2.58 mmol) in dichloromethane (5 mL) was added thionylchloride (922 mg, 564 μL, 7.75 mmol) and N,N-dimethyl formamide (20 μL).The reaction mixture was stirred at room temperature for 30 minutesbefore it was evaporated to dryness to yield3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoylchloride as a yellow solid.

Step b: 2-(Dimethylamino)ethyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoate

To N,N-dimethylethanol amine (921 mg, 1.04 mL, 10.3 mmol) indichloromethane (5 mL) was added a solution of3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoylchloride in dichloromethane (5 mL). The reaction mixture was stirred atroom temperature overnight. The mixture was diluted with DCM (20 mL) andwas washed with 1N HCl (20 mL) and saturated NaHCO₃ (20 mL). Theorganics were dried over Na₂SO₄ and evaporated to yield2-(dimethylamino)ethyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoate(1.30 g, 90%). ESI-MS m/z calc. 559.6. found 560.3 (M+1)⁺. Retentiontime 1.72 minutes.

Step c: 2-(3-(3-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoyloxy)-N,N,N-trimethylethanaminiumchloride

To 2-(dimethylamino)ethyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoate(478 mg, 0.855 mmol) in acetone (10 mL) was added iodomethane (1.00 mL,16.1 mmol). After stirring for 1.5 h, the white precipitate that hadformed was collected by vacuum filtration and was washed with coldacetone to yield a solid as the ammonium iodide salt. The material wasdissolved in 1.25 M HCl in methanol (1.91 mL, 2.39 mmol) and heated at60° C. for 1 h. The reaction was cooled to room temperature and acetonewas added to yield a precipitate. The precipitate was dissolved in DCMand was washed with 1 N HCl (2×10 mL). The organics were dried overNa₂SO₄ and evaporated to give an oil which upon re-evaporating withDCM/hexane gave2-(3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoyloxy)-N,N,N-trimethylethanaminiumchloride (358 mg, 93%). ESI-MS m/z calc. 610.2. found 610.3 (M+1)⁺.Retention time 1.79 minutes.

Preparation:N-(1-(3-(Acetamidomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

Step a: tert-Butyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzylcarbamate

tert-Butyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzylcarbamatewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 3-((tert-butoxycarbonylamino)methyl)phenylboronic acid. ESI-MS m/zcalc. 573.2. found 574.5 (M+1)⁺. Retention time 2.26 minutes. ¹H NMR(400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.43 (s, 1H), 7.98 (d, J=8.4 Hz, 1H),7.83 (d, J=8.6 Hz, 1H), 7.74-7.70 (m, 1H), 7.60 (s, 1H), 7.50-7.37 (m,8H), 4.20 (d, J=6.3 Hz, 2H), 1.57-1.54 (m, 2H), 1.37 (s, 9H), 1.22-1.19(m, 2H).

Step b:N-(1-(3-(Aminomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

TFA (500 μL) was added to a solution of tert-butyl3-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzylcarbamate(115 mg, 0.200 mmol) in CH₂Cl₂ (1.5 mL). The reaction was stirred atroom temperature for 1 hour. The reaction was diluted with CH₂Cl₂ and 1NNaOH was added until the mixture become basic. The organic layer wasdried over MgSO₄, filtered and concentrated to yieldN-(1-(3-(Aminomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideas a white solid (95 mg, 99%). ESI-MS m/z calc. 473.2. found 474.2(M+1)⁺. Retention time 1.62 minutes.

Step c:N-(1-(3-(Acetamidomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

To a solution ofN-(1-(3-(aminomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide(47 mg, 0.1 mmol) and Et₃N (28 μL, 0.2 mmol) in DMF (2 mL) was addedacetyl chloride (7.1 μL, 0.1 mmol). The reaction was stirred at roomtemperature for 10 minutes, then filtered and purified by reverse-phaseHPLC to yield the product,N-(1-(3-(acetamidomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide.ESI-MS m/z calc. 515.2. found 516.5 (M+1)⁺. Retention time 1.97 minutes.¹H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.43-8.40 (m, 2H), 7.98 (d,J=8.2 Hz, 1H), 7.83 (d, J=8.4 Hz, 1H), 7.74-7.70 (m, 1H), 7.61 (d, J=1.1Hz, 1H), 7.50-7.38 (m, 7H), 4.33 (d, J=6.0 Hz, 2H), 1.85 (s, 3H),1.57-1.55 (m, 2H), 1.22-1.19 (m, 2H).

Preparation:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(3-(methylsulfonamidomethyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(3-(methylsulfonamidomethyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-(3-(aminomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand methanesulfonyl chloride. ESI-MS m/z calc. 551.1. found 552.3(M+1)⁺. Retention time 2.06 minutes. ¹H NMR (400 MHz, DMSO-d6) δ 9.08(s, 1H), 8.43 (s, 1H), 7.98 (d, J=8.4 Hz, 1H), 7.84 (d, J=8.9 Hz, 1H),7.75-7.71 (m, 1H), 7.64-7.60 (m, 2H), 7.54-7.40 (m, 7H), 4.25 (d, J=6.3Hz, 2H), 2.89 (s, 3H), 1.57-1.55 (m, 2H), 1.22-1.19 (m, 2H).

Preparation:N-(1-(4-(Acetamidomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

Step a: tert-Butyl4-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzylcarbamate

tert-Butyl4-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzylcarbamatewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand 4-((tert-butoxycarbonylamino)methyl)phenylboronic acid. ESI-MS m/zcalc. 573.2. found 574.3 (M+1)⁺. Retention time 2.26 minutes. ¹H NMR(400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.40 (s, 1H), 7.97 (d, J=8.2 Hz, 1H),7.86 (d, J=8.6 Hz, 1H), 7.74-7.70 (m, 1H), 7.61 (d, J=1.2 Hz, 1H),7.54-7.38 (m, 8H), 4.22 (d, J=5.9 Hz, 2H), 1.57-1.55 (m, 2H), 1.41 (s,9H), 1.22-1.20 (m, 2H).

Step b:N-(1-(4-(Aminomethyl)phenyl]isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

N-(1-(4-(Aminomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamidewas made by the procedure shown above starting from tert-butyl4-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzylcarbamate.ESI-MS m/z calc. 473.2. found 474.2 (M+1)⁺. Retention time 1.61 minutes.

Step c:N-(1-(4-(Acetamidomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

N-(1-(4-(Acetamidomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-(4-(aminomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand acetyl chloride. ESI-MS m/z calc. 515.2. found 516.5 (M+1)⁺.Retention time 1.96 minutes. ¹H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H),8.46-8.42 (m, 2H), 7.98 (d, J=8.3 Hz, 1H), 7.87 (d, J=8.5 Hz, 1H),7.75-7.71 (m, 1H), 7.61 (d, J=1.1 Hz, 1H), 7.55-7.39 (m, 7H), 4.35 (d,J=5.9 Hz, 2H), 1.91 (s, 3H), 1.59-1.56 (m, 2H), 1.23-1.20 (m, 2H).

Preparation:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-(methylsulfonamidomethyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(4-(methylsulfonamidomethyl)phenyl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-(4-(aminomethyl)phenyl)isoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamideand methanesulfonyl chloride. ESI-MS m/z calc. 551.1. found 552.3(M+1)⁺. Retention time 2.06 minutes. ¹H NMR (400 MHz, DMSO-d6) δ 9.00(s, 1H), 8.41 (s, 1H), 7.98 (d, J=8.3 Hz, 1H), 7.85 (d, J=8.5 Hz, 1H),7.74-7.70 (m, 1H), 7.66 (t, J=6.5 Hz, 1H), 7.61 (d, J=1.2 Hz, 1H), 7.57(d, J=8.1 Hz, 2H), 7.52-7.47 (m, 3H), 7.44-7.38 (m, 2H), 4.26 (d, J=6.3Hz, 2H), 2.92 (s, 3H), 1.57-1.55 (m, 2H), 1.22-1.20 (m, 2H).

Preparation:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(6-oxo-1,6-dihydropyridin-3-yl)isoquinolin-3-yl)cyclopropanecarboxamide

Step a:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(6-methoxypyridin-3-yl)isoquinolin-3-yl)cyclopropanecarboxamide

N-(1-Bromoisoquinolin-3-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide(268 mg, 0.600 mmol) was dissolved in 6 mL of 1,2-dimethoxyethane (DME)in a microwave reactor tube. 6-Methoxypyridin-3-ylboronic acid (119 mg,0.780 mmol), 0.6 mL of an aqueous 2 M potassium carbonate solution, andtetrakis(triphenylphospine)palladium(0) (Pd(PPh₃)₄, 34.7 mg, 0.0300mmol) were added and the reaction mixture was heated at 120° C. in amicrowave reactor for 20 minutes. The resulting material was cooled toroom temperature, filtered, and the layers were separated. The crudeproduct was evaporated to dryness and then purified on silica gelutilizing a gradient of 0-50% ethyl acetate in hexanes to yield1-(2,2-difluorobenzo-[d][1,3]dioxol-5-yl)-N-(1-(6-methoxypyridin-3-yl)isoquinolin-3-yl)cyclopropane-carboxamide(204 mg, 71%). ESI-MS m/z calc. 475.1. found; 476.3 (M+1)⁺ Retentiontime 2.31 minutes.

Step b:1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(6-oxo-1,6-dihydropyridin-3-yl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(6-methoxypyridin-3-yl)isoquinolin-3-yl)cyclopropanecarboxamide(100 mg, 0.210 mmol) was dissolved in a mixture of 1.2 mL of 1,4-dioxaneand 0.6 mL of 4M aqueous hydrochloric acid. The solution was heated at90° C. for 2 hours. The crude reaction mixture was quenched with oneequivalent of triethylamine and was then evaporated to dryness. Thecrude product was partitioned between dichloromethane and water. Theorganic layer was separated, dried over sodium sulfate, and a portion ofthe material was purified on 12 g of silica gel utilizing a gradient of0-10% methanol in dichloromethane to yield1-(2,2-difluorobenzo-[d][1,3]dioxol-5-yl)-N-(1-(6-oxo-1,6-dihydropyridin-3-yl)isoquinolin-3-yl)cyclopropane-carboxamideas a white solid. ESI-MS m/z calc. 461.1. found 461.9 (M+1)⁺. Retentiontime 1.67 minutes.

Preparation:1-(4-methoxyphenyl)-N-(1-morpholinoisoquinolin-3-yl)cyclopropanecarboxamide

N-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropane-carboxamide(39.7 mg, 0.1 mmol) was dissolved in 1,4-dioxane (1.0 mL) in a microwavetube. Morpholine (26.14 μL, 0.3 mmol) was added and the reaction wasstirred and heated at 170° C. for eighteen hours. The solvent wasevaporated. The crude product was dissolved in DMSO (1 mL), filtered andpurified by reverse phase preparative HPLC to yield1-(4-methoxyphenyl)-N-(1-morpholinoisoquinolin-3-yl)cyclopropanecarboxamideas a TFA salt. ESI-MS m/z calc. 403.19. found 404.5 (M+1)⁺. Retentiontime 1.95 minutes. ¹H NMR (400.0 MHz, DMSO) d 8.01-7.98 (m, 2H), 7.93(s, 1H), 7.80 (d, J=8.2 Hz, 1H), 7.64-7.60 (m, 1H), 7.49-7.41 (m, 3H),7.05-7.01 (m, 2H), 3.80-3.78 (m, 7H), 3.17 (t, J=4.4 Hz, 4H), 1.51 (dd,J=3.8, 6.7 Hz, 2H) and 1.13 (dd, J=3.9, 6.8 Hz, 2H) ppm.

Preparation:1-(4-methoxyphenyl)-N-(1-(4-methylpiperazin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(4-methoxyphenyl)-N-(1-(4-methylpiperazin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropane-carboxamideand 1-methylpiperazine.

Preparation:1-(4-methylphenyl)-N-(1-(3-oxo-4-phenylpiperazin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(4-methoxyphenyl)-N-(1-(3-oxo-4-phenylpiperazin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamideand 1-phenylpiperazin-2-one.

Preparation:1-(4-methoxyphenyl)-N-(1-(3-oxopiperazin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(4-methoxyphenyl)-N-(1-(3-oxopiperazin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamideand piperazin-2-one.

Preparation:1-(4-methoxyphenyl)-N-(1-(4-phenylpiperidin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(4-methoxyphenyl)-N-(1-(4-phenylpiperidin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamideand 4-phenylpiperidine.

Preparation:1-(4-methoxyphenyl)-N-(1-(piperidin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamide

1-(4-methoxyphenyl)-N-(1-(piperidin-1-yl)isoquinolin-3-yl)cyclopropanecarboxamidewas made by the procedure shown above starting fromN-(1-bromoisoquinolin-3-yl)-1-(4-methoxyphenyl)cyclopropanecarboxamideand piperidine.

Physical data for examples of the invention are given in Table 2.

TABLE 2 Com- pound LC/MS LC/RT No. [M + H]⁺ min NMR 1 552.3 2.06 H NMR(400 MHz, DMSO) 9.00 (s, 1H), 8.41 (s, 1H), 7.98 (d, J = 8.3 Hz, 1H),7.85 (d, J = 8.5 Hz, 1H), 7.74-7.70 (m, 1H), 7.66 (t, J = 6.5 Hz, 1H),7.61 (d, J = 1.2 Hz, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.52-7.47 (m, 3H),7.44- 7.38 (m, 2H), 4.26 (d, J = 6.3 Hz, 2H), 2.92 (s, 3H), 1.57-1.55(m, 2H), 1.22-1.20 (m, 2H) 2 485.5 2.01 H NMR (400 MHz, DMSO) 9.05 (d, J= 9.9 Hz, 1H), 8.41 (s, 1H), 8.19 (s, 1H), 7.99-7.92 (m, 3H), 7.74-7.67(m, 2H), 7.63-7.56 (m, 2H), 7.51-7.38 (m, 3H), 1.58-1.55 (m, 2H),1.22-1.20 (m, 2H) 3 516.5 1.96 H NMR (400 MHz, DMSO) 9.08 (s, 1H), 8.43(s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.75-7.71(m, 1H), 7.64-7.60 (m, 2H), 7.54-7.40 (m, 7H), 4.25 (d, J = 6.3 Hz, 2H),2.89 (s, 3H), 1.57-1.55 (m, 2H), 1.22-1.19 (m, 2H) 4 445.3 2.45 5 489.52.12 H NMR (400 MHz, DMSO) 9.00 (s, 1H), 8.41 (s, 1H), 7.98 (d, J = 8.3Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.74-7.70 (m, 1H), 7.66 (t, J = 6.5Hz, 1H), 7.61 (d, J = 1 .2 Hz, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.52-7.47(m, 3H), 7.44-7.38 (m, 2H), 4.26 (d, J = 6.3 Hz, 2H), 2.92 (s, 3H),1.57-1.55 (m, 2H), 1.22-1.20 (m, 2H) 6 488.3 1.92 H NMR (400 MHz, DMSO)9.10 (s, 1H), 8.46 (s, 1H), 8.11-7.98 (m, 4H), 7.84 (d, J = 8.5 Hz, 1H),7.78-7.72 (m, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 1.1 Hz, 1H),7.53- 7.49 (m, 2H), 7.43-7.38 (m, 2H), 1.58-1.56 (m, 2H), 1.23-1.20 (m,2H) 7 498.3 2.12 8 489.3 2.12 9 552.3 2.06 10 516.5 1.97 H NMR (400 MHz,DMSO) 9.06 (s, 1H), 8.43-8.40 (m, 2H), 7.98 (d, J = 8.2 Hz, 1H), 7.83(d, J = 8.4 Hz, 1H), 7.74-7.70 (m, 1H), 7.61 (d, J = 1.1 Hz, 1H),7.50-7.38 (m, 7H), 4.33 (d, J = 6.0 Hz, 2H), 1.85 (s, 3H), 1.57- 1.55(m, 2H), 1.22-1.19 (m, 2H) 11 524.3 2.03 12 417.5 1.46 13 524.3 2.02 14493.7 1.99 15 500.3 1.93 H NMR (400 MHz, DMSO) 8.93 (s, 1H), 8.46-8.42(m, 2H), 7.98 (d, J = 8.3 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.75-7.71(m, 1H), 7.61 (d, J = 1.1 Hz, 1H), 7.55-7.39 (m, 7H), 4.35 (d, J = 5.9Hz, 2H), 1.91 (s, 3H), 1.59- 1.56 (m, 2H), 1.23-1.20 (m, 2H) 16 484.52.08 H NMR (400 MHz, DMSO) 11.30 (s, 1H), 8.94 (s, 1H), 8.37 (s, 1H),7.99-7.94 (m, 2H), 7.76 (s, 1H), 7.70 (t, J = 7.4 Hz, 1H), 7.62 (s, 1H),7.54-7.42 (m, 5H), 7.31 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 1.58-1.55 (m,2H), 1.22- 1.20 (m, 2H) 17 461.9 1.67 H NMR (400 MHz, DMSO) 9.08 (s,1H), 8.47 (s, 1H), 8.02-7.97 (m, 3H), 7.83-7.73 (m, 4H), 7.61 (d, J =1.3 Hz, 1H), 7.54-7.38 (m, 5H), 1.58-1.56 (m, 2H), 1.23-1.20 (m, 2H) 18404.5 1.94 19 560.3 1.74 20 478.5 2.35 21 417.5 1.63 22 488.3 1.91 H NMR(400 MHz, DMSO) 9.14 (s, 1H), 8.45 (s, 1H), 8.08-7.99 (m, 4H), 7.82 (d,J = 8.4 Hz, 1H), 7.76-7.72 (m, 2H), 7.64-7.60 (m, 2H), 7.53-7.38 (m,4H), 1.58-1.55 (m, 2H), 1.22-1.19 (m, 2H) 23 402.7 1.97 24 571.3 1.68 25476.3 2.31 26 552.3 2.12 H NMR (400 MHz, DMSO) 9.22 (s, 1H), 8.48 (s,1H), 8.03-7.96 (m, 3H), 7.83-7.73 (m, 4H), 7.60-7.37 (m, 6H), 1.58-1.55(m, 2H), 1.22-1.19 (m, 2H) 27 475.3 2.02 H NMR (400 MHz, DMSO) 8.98 (s,1H), 8.41 (s, 1H), 8.00-7.96 (m, 1H), 7.88-7.84 (m, 1H), 7.72 (t, J =7.1 Hz, 1H), 7.64-7.38 (m, 8H), 5.54 (s, 1H), 4.61 (s, 2H), 1.58-1.55(m, 2H), 1.22-1.20 (m, 2H) 28 574.5 1.77 H NMR (400 MHz, DMSO) 9.19 (s,1H), 8.50 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.79 (s, 1H), 7.76-7.72 (m,2H), 7.59 (d, J = 0.7 Hz, 1H), 7.54-7.37 (m, 6H), 2.48 (d, J = 4.9 Hz,3H), 2.02 (s, 3H), 1.57-1.56 (m, 2H), 1.21- 1.19 (m, 2H)

Assays

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

1. Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells weresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 μM forskolin and theCFTR potentiator, genistein (20 μM), were added along with Cl⁻-freemedium to each well. The addition of Cl⁻-free medium promoted Cl⁻ effluxin response to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

2. Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 μM forskolin was added toactivate ΔF508-CFTR. The extracellular Cl⁻ concentration following bothadditions was 28 mM, which promoted CF efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization was opticallymonitored using the FRET-based voltage-sensor dyes.

3. Solutions

-   -   Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1,        HEPES 10, pH 7.4 with NaOH.    -   Chloride-free bath solution: Chloride salts in Bath Solution #1        are substituted with gluconate salts.    -   CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored        at −20° C.    -   DiSBAC₂(3): Prepared as a 10 mM stock in DMSO and stored at −20°        C.

4. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hours.

Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds

1. Using Chamber Assay

Using chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulatorsidentified in the optical assays. FRT^(ΔF508-CFTR) epithelial cellsgrown on Costar Snapwell cell culture inserts were mounted in an Usingchamber (Physiologic Instruments, Inc., San Diego, Calif.), and themonolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, IA, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 KΩ/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through ΔF508-CFTR expressed in the apical membrane. The I_(SC)was digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

2. Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(SC) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

3. Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30 minafter nystatin permeabilization. Forskolin (10 μM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

4. Solutions

-   -   Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂        (1.2), K₂HPO₄ (2.4), KHPO₄ (0.6),        N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)        (10), and dextrose (10). The solution was titrated to pH 7.4        with NaOH.    -   Apical solution (in mM): Same as basolateral solution with NaCl        replaced with Na Gluconate (135).

5. Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Using chamber experiments for theputative ΔF508-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16-48 hrsto correct for the ΔF508-CFTR. To determine the activity of correctionscompounds, the cells were incubated at 27° C. or 37° C. with and withoutthe compounds for 24 hours.

6. Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature- and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for a(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance >20 GΩ and a series resistance <15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 μl of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system.

7. Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 μM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

8. Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

9. Solutions

-   -   Intracellular solution (in mM): Cs-aspartate (90), CsCl (50),        MgCl₂ (1), HEPES (10), and 240 μg/ml amphotericin-B (pH adjusted        to 7.35 with CsOH).    -   Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl        (150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35        with HCl).

10. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1×pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

11. Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5-8 MS2 when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1-2 sec. To maintainΔF508-CFTR activity during the rapid perifusion, the nonspecificphosphatase inhibitor F⁻ (10 mM NaF) was added to the bath solution.Under these recording conditions, channel activity remained constantthroughout the duration of the patch recording (up to 60 min) Currentsproduced by positive charge moving from the intra- to extracellularsolutions (anions moving in the opposite direction) are shown aspositive currents. The pipette potential (V_(p)) was maintained at 80mV.

Channel activity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

12. Solutions

-   -   Extracellular solution (in mM): NMDG (150), aspartic acid (150),        CaCl₂ (5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with        Tris base).    -   Intracellular solution (in mM): NMDG-Cl (150), MgCl₂ (2), EGTA        (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with        HCl).

13. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

The exemplified compounds of Table 1 have an activity with a range ofabout 10 nM and 10 μM as measured using the assays describedhereinabove. The exemplified compounds of Table 1 are found to besufficiently efficacious as measured using the assays describedhereinabove.

TABLE 3 Cmpd. No. Binned EC50 Binned Max Efficacy 1 + ++ 2 +++ +++ 3 +++++ 4 +++ +++ 5 +++ +++ 6 +++ +++ 7 +++ +++ 8 +++ +++ 9 +++ +++ 10 ++++++ 11 +++ ++ 12 +++ +++ 13 +++ +++ 14 +++ ++ 15 +++ ++ 16 +++ +++ 17+++ ++ 18 +++ +++ 19 +++ ++ 20 +++ +++ 21 +++ ++ 22 +++ +++ 23 +++ +++24 +++ ++ 25 +++ +++ 26 +++ ++ 27 +++ +++ 28 +++ ++ IC50/EC50 Bins: +++<= 2.0 μM < ++ <= 5.0 μM < + PercentActivity Bins: + <= 25.0 < ++ <=100.0 < +++

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating or lessening the severity ofa disease in a patient, wherein the disease is cystic fibrosiscomprising the step of administering to the patient an effective amountof a compound

wherein the patient has a defective gene that causes a deletion ofphenylalanine at position 508 of the cystic fibrosis transmembraneconductance regulator amino acid sequence.
 2. The method of claim 1,wherein the patient has two copies of the defective gene.
 3. A method oftreating or lessening the severity of cystic fibrosis in a patient,wherein the patient has a defective gene that causes a deletion ofphenylalanine at position 508 of the cystic fibrosis transmembraneconductance regulator amino acid sequence, said method comprising thestep of administering to said patient an effective amount of acomposition comprising: i) the compound:

or a pharmaceutically acceptable salt thereof; and ii) apharmaceutically acceptable carrier.
 4. The method of claim 3, whereinthe patient has two copies of the defective gene.
 5. The method of claim3 or 4, wherein the composition further comprises a mucolytic agent, abronchodialator, an antibiotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator, or a nutritional agent. 6.The method of claim 3 or 4, wherein the composition further comprises aCFTR modulator.